Use of HMGB fragments as anti-inflammatory agents

ABSTRACT

Compositions and methods are disclosed for inhibiting the release of a proinflammatory cytokine from a vertebrate cell, and for inhibiting an inflammatory cytokine cascade in a patient. The compositions comprise a vertebrate HMGB A box, and an antibody preparation that specifically binds to a vertebrate HMGB B box. The methods comprise treating a cell or a patient with sufficient amounts of the composition to inhibit the release of the proinflammatory cytokine, or to inhibit the inflammatory cytokine cascade.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/300,072, filed on Nov. 20, 2002, now U.S. Pat. No. 7,304,034, whichis a continuation-in-part of U.S. application Ser. No. 10/147,447, filedMay 15, 2002, now abandoned, which claims the benefit of U.S.Provisional Application No. 60/291,034, filed on May 15, 2001.

The entire teachings of the above applications are incorporated hereinby reference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by a grant RO1 GM062508 from the National Institutes of Health. The Government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

Inflammation is often induced by proinflammatory cytokines, such astumor necrosis factor (TNF), interleukin (IL)-1α, IL-1β, IL-6,platelet-activating factor (PAF), macrophage migration inhibitory factor(MIF), and other compounds. These proinflammatory cytokines are producedby several different cell types, most importantly immune cells (forexample, monocytes, macrophages and neutrophils), but also non-immunecells such as fibroblasts, osteoblasts, smooth muscle cells, epithelialcells, and neurons. These proinflammatory cytokines contribute tovarious disorders during the early stages of an inflammatory cytokinecascade.

Inflammatory cytokine cascades contribute to deleteriouscharacteristics, including inflammation and apoptosis, of numerousdisorders. Included are disorders characterized by both localized andsystemic reactions, including, without limitation, diseases involvingthe gastrointestinal tract and associated tissues (such as appendicitis,peptic, gastric and duodenal ulcers, peritonitis, pancreatitis,ulcerative, pseudomembranous, acute and ischemic colitis,diverticulitis, epiglottitis, achalasia, cholangitis, cholecystitis,coeliac disease, hepatitis, Crohn's disease, enteritis, and Whipple'sdisease); systemic or local inflammatory diseases and conditions (suchas asthma, allergy, anaphylactic shock, immune complex disease, organischemia, reperfusion injury, organ necrosis, hay fever, sepsis,septicemia, endotoxic shock, cachexia, hyperpyrexia, eosinophilicgranuloma, granulomatosis, and sarcoidosis); diseases involving theurogenital system and associated tissues (such as septic abortion,epididymitis, vaginitis, prostatitis, and urethritis); diseasesinvolving the respiratory system and associated tissues (such asbronchitis, emphysema, rhinitis, cystic fibrosis, pneumonitis, adultrespiratory distress syndrome,pneumoultramicroscopicsilicovolcanoconiosis, alvealitis, bronchiolitis,pharyngitis, pleurisy, and sinusitis); diseases arising from infectionby various viruses (such as influenza, respiratory syncytial virus, HIV,hepatitis B virus, hepatitis C virus and herpes), bacteria (such asdisseminated bacteremia, Dengue fever), fungi (such as candidiasis) andprotozoal and multicellular parasites (such as malaria, filariasis,amebiasis, and hydatid cysts); dermatological diseases and conditions ofthe skin (such as burns, dermatitis, dermatomyositis, sunburn, urticariawarts, and wheals); diseases involving the cardiovascular system andassociated tissues (such as vasulitis, angiitis, endocarditis,arteritis, atherosclerosis, thrombophlebitis, pericarditis, congestiveheart failure, myocarditis, myocardial ischemia, periarteritis nodosa,and rheumatic fever); diseases involving the central or peripheralnervous system and associated tissues (such as Alzheimer's disease,meningitis, encephalitis, multiple sclerosis, cerebral infarction,cerebral embolism, Guillame-Barre syndrome, neuritis, neuralgia, spinalcord injury, paralysis, and uveitis); diseases of the bones, joints,muscles and connective tissues (such as the various arthritides andarthralgias, osteomyelitis, fasciitis, Paget's disease, gout,periodontal disease, rheumatoid arthritis, and synovitis); otherautoimmune and inflammatory disorders (such as myasthenia gravis,thryoiditis, systemic lupus erythematosus, Goodpasture's syndrome,Behcets's syndrome, allograft rejection, graft-versus-host disease, TypeI diabetes, ankylosing spondylitis, Berger's disease, Type I diabetes,ankylosing spondylitis, Berger's disease, and Retier's syndrome); aswell as various cancers, tumors and proliferative disorders (such asHodgkins disease); and, in any case the inflammatory or immune hostresponse to any primary disease.

The early proinflammatory cytokines (e.g., TNF, IL-1, etc.) mediateinflammation, and induce the late release of high mobility group-1(HMG1) (also known as HMG-1 and HMGB1), a protein that accumulates inserum and mediates delayed lethality and further induction of earlyproinflammatory cytokines.

HMGB1 was first identified as the founding member of a family ofDNA-binding proteins termed high mobility group (HMG) that are criticalfor DNA structure and stability. It was identified nearly 40 years agoas a ubiquitously expressed nuclear protein that binds double-strandedDNA without sequence specificity.

HMGB1 binding bends DNA to promote formation and stability ofnucleoprotein complexes that facilitate gene transcription ofglucocorticoid receptors and RAG recombinase. The HMGB1 molecule hasthree domains: two DNA binding motifs termed HMGB A and HMGB B boxes,and an acidic carboxyl terminus. The two HMGB boxes are highly conserved80 amino acid, L-shaped domains. HMGB boxes are also expressed in othertranscription factors including the RNA polymerase I transcriptionfactor human upstream-binding factor and lymphoid-specific factor.

Recent evidence has implicated HMGB1 as a cytokine mediator of delayedlethality in endotoxemia. That work demonstrated that bacterialendotoxin (lipopolysaccharide (LPS)) activates monocytes/macrophages torelease HMGB1 as a late response to activation, resulting in elevatedserum HMGB1 levels that are toxic. Antibodies against HMGB1 preventlethality of endotoxin even when antibody administration is delayeduntil after the early cytokine response. Like other proinflammatorycytokines, HMGB1 is a potent activator of monocytes. Intratrachealapplication of HMGB1 causes acute lung injury, and anti-HMGB1 antibodiesprotect against endotoxin-induced lung edema. Serum HMGB1 levels areelevated in critically ill patients with sepsis or hemorrhagic shock,and levels are significantly higher in non-survivors as compared tosurvivors.

HMGB1 has also been implicated as a ligand for RAGE, a multi-ligandreceptor of the immunoglobulin superfamily. RAGE is expressed onendothelial cells, smooth muscle cells, monocytes, and nerves, andligand interaction transduces signals through MAP kinase, P21 ras, andNF-κB. The delayed kinetics of HMGB1 appearance during endotoxemia makesit a potentially good therapeutic target, but little is known about themolecular basis of HMGB1 signaling and toxicity.

Therefore, it would be useful to identify characteristics of HMGB1proinflammatory activity, particularly the active domain(s) responsiblefor this activity, and any inhibitory effects of other domains.

SUMMARY OF THE INVENTION

The present invention is based on the discoveries that (1) the HMGB Abox serves as a competitive inhibitor of HMGB proinflammatory action,and (2) the HMGB B box has the predominant proinflammatory activity ofHMG.

Accordingly, the present invention is directed to a polypeptidecomprising a vertebrate HMGB A box or a biologically active fragmentthereof or a non-naturally occurring HMGB A box or a biologically activefragment thereof. The HMGB A box or these embodiments can inhibitrelease of a proinflammatory cytokine from a vertebrate cell treatedwith HMG. The HMGB A box is preferably a mammalian HMGB A box, morepreferably, a mammalian HMGB1 A box, for example, a human HMGB1 A box,and most preferably, the HMGB1 A box comprising or consisting of thesequence of SEQ ID NO:4, SEQ ID NO:22, or SEQ ID NO:57. In a preferredembodiment, the vertebrate cell is a mammalian macrophage. The presentinvention also encompasses vectors encoding these polypeptides.

In other embodiments, the invention is directed to a compositioncomprising the HMGB A box polypeptide or a biologically active fragmentthereof described above in a pharmaceutically acceptable excipient. Inthese embodiments, the composition can inhibit a condition characterizedby activation of an inflammatory cytokine cascade. The composition canfurther comprise an antagonist of an early sepsis mediator. Theantagonist of an early sepsis mediator is preferably an antagonist of acytokine selected from the group consisting of TNF, IL-1α, IL-1β, MIFand IL-6, more preferably, an antibody to TNF or MIF, or an IL-1receptor antagonist.

In these embodiments, the condition is preferably selected from thegroup consisting of appendicitis, peptic, gastric and duodenal ulcers,peritonitis, pancreatitis, ulcerative, pseudomembranous, acute andischemic colitis, diverticulitis, epiglottitis, achalasia, cholangitis,cholecystitis, hepatitis, Crohn's disease, enteritis, Whipple's disease,asthma, allergy, anaphylactic shock, immune complex disease, organischemia, reperfusion injury, organ necrosis, hay fever, sepsis,septicemia, endotoxic shock, cachexia, hyperpyrexia, eosinophilicgranuloma, granulomatosis, sarcoidosis, septic abortion, epididymitis,vaginitis, prostatitis, urethritis, bronchitis, emphysema, rhinitis,cystic fibrosis, pneumonitis,pneumoultramicroscopicsilicovolcanoconiosis, alvealitis, bronchiolitis,pharyngitis, pleurisy, sinusitis, influenza, respiratory syncytial virusinfection, herpes infection, HIV infection, hepatitis B virus infection,hepatitis C virus infection, disseminated bacteremia, Dengue fever,candidiasis, malaria, filariasis, amebiasis, hydatid cysts, burns,dermatitis, dermatomyositis, sunburn, urticaria, warts, wheals,vasulitis, angiitis, endocarditis, arteritis, atherosclerosis,thrombophlebitis, pericarditis, myocarditis, myocardial ischemia,periarteritis nodosa, rheumatic fever, Alzheimer's disease, coeliacdisease, congestive heart failure, adult respiratory distress syndrome,meningitis, encephalitis, multiple sclerosis, cerebral infarction,cerebral embolism, Guillame-Barre syndrome, neuritis, neuralgia, spinalcord injury, paralysis, uveitis, arthritides, arthralgias,osteomyelitis, fasciitis, Paget's disease, gout, periodontal disease,rheumatoid arthritis, synovitis, myasthenia gravis, thryoiditis,systemic lupus erythematosus, Goodpasture's syndrome, Behcets'ssyndrome, allograft rejection, graft-versus-host disease, Type Idiabetes, ankylosing spondylitis, Berger's disease, Type I diabetes,ankylosing spondylitis, Berger's disease, Retier's syndrome, andHodgkins disease. More preferably, the condition is selected from thegroup consisting of appendicitis, peptic, gastric and duodenal ulcers,peritonitis, pancreatitis, ulcerative, pseudomembranous, acute andischemic colitis, hepatitis, Crohn's disease, asthma, allergy,anaphylactic shock, organ ischemia, reperfusion injury, organ necrosis,hay fever, sepsis, septicemia, endotoxic shock, cachexia, septicabortion, disseminated bacteremia, burns, Alzheimer's disease, coeliacdisease, congestive heart failure, adult respiratory distress syndrome,cerebral infarction, cerebral embolism, spinal cord injury, paralysis,allograft rejection and graft-versus-host disease; most preferably, thecondition is endotoxic shock or allograft rejection. When the conditionis allograft rejection, the composition can further comprise animmunosuppressant used to inhibit allograft rejection, preferablycyclosporin.

In additional embodiments, the invention is directed to a purifiedpreparation of antibodies that specifically bind to a vertebrate highmobility group protein (HMG) B box but do not specifically bind to non-Bbox epitopes of HMG. In these embodiments, the antibodies can inhibit abiological activity of an HMGB B box polypeptide, for example, therelease of a proinflammatory cytokine from a vertebrate cell treatedwith HMG. In preferred embodiments, the HMGB B box is a mammalian HMGB Bbox, for example, a human HMGB B box, more preferably an HMGB1 B box,most preferably the HMGB1 B box with the amino acid sequence of SEQ IDNO:5, SEQ ID NO:20, or SEQ ID NO:58. In another embodiment, theantibodies bind a specific polypeptide sequence of the HMGB1 B box,comprising amino acids 1-20 of SEQ ID NO:20 (SEQ ID NO:23), orcomprising amino acids 1-20 of SEQ ID NO:5 (SEQ ID NO:16), or consistingof amino acids 1-20 of SEQ ID NO:20 (SEQ ID NO:23), or consisting ofamino acids 1-20 of SEQ ID NO:5 (SEQ ID NO:23). The vertebrate cell isalso preferably a mammalian macrophage. In some embodiments, theantibodies are preferably humanized.

In additional embodiments, the invention is directed to a compositioncomprising any of the antibody preparations described above, in apharmaceutically acceptable excipient. In these embodiments, thecomposition can inhibit a condition characterized by activation of aninflammatory cytokine cascade. These compositions can also usefullycomprise an antagonist of an early sepsis mediator, as previouslydescribed. The preferred conditions useful for treatment with thesecompositions are those mediated or characterized by activation of aninflammatory cytokine cascade, for example, those conditions asenumerated with the A box compositions previously described.

Additionally, the present invention is directed to a polypeptidecomprising a vertebrate HMGB B box or a biologically active fragmentthereof or a non-naturally occurring HMGB B box or biologically activefragment thereof, but not comprising a full length HMGB protein. Inthese embodiments, the polypeptide can cause release of aproinflammatory cytokine from a vertebrate cell. The polypeptide ofthese embodiments is preferably an HMGB B box, more preferably an HMGB1B box, most preferably the HMGB1 B box with the amino acid sequencegiven as SEQ ID NO:5, SEQ ID NO:20, or SEQ ID NO:58. In anotherembodiment, the HMGB B box fragment comprises the sequence of SEQ IDNO:16 or SEQ ID NO:23 or consists of the sequence of SEQ ID NO:16 or SEQID NO:23. In a preferred embodiment, the vertebrate cell is a mammalianmacrophage. The present invention also encompasses vectors encodingthese polypeptides.

The present invention is also directed to a method of inhibiting releaseof a proinflammatory cytokine from a mammalian cell. The methodcomprises treating the cell with either the A box or A box biologicallyactive fragment polypeptide composition described above or the B box orB box biologically active fragment antibody compositions describedabove, in an amount sufficient to inhibit release of the proinflammatorycytokine from the cell. In these embodiments, the cell is preferably amacrophage. In addition, the proinflammatory cytokine is preferablyselected from the group consisting of TNF, IL-1α, IL-1β, MIF and IL-6.More preferably the cell is a macrophage and the proinflammatorycytokine is preferably selected from the group consisting of TNF, IL-1α,IL-1β, MIF and IL-6. The methods preferably treat a cell in a patientsuffering from, or at risk for, a condition characterized by activationof the inflammatory cytokine cascade. Preferred conditions have beenenumerated previously.

In related embodiments, the present invention is directed to a method oftreating a condition in a patient characterized by activation of aninflammatory cytokine cascade. The method comprises administering to thepatient any of the A box or A box biologically active fragmentpolypeptide compositions or the B box or B box biologically activefragment antibody compositions described above in an amount sufficientto inhibit the inflammatory cytokine cascade. Preferred conditions havealready been enumerated.

Additional embodiments are directed to a method of stimulating therelease of a proinflammatory cytokine from a cell. The method comprisestreating the cell with the B box polypeptide or a biologically activefragment thereof, or the vector of the B box polypeptide or B boxbiologically active fragment previously described in an amountsufficient to stimulate the release of the proinflammatory cytokine. Inrelated embodiments, the invention is directed to a method for effectingweight loss or treating obesity in a patient. The method comprisesadministering to the patient an effective amount of the HMGB B boxpolypeptide or a biologically active fragment thereof to the patient. Inone embodiment, the HMGB B box polypeptide or a biologically activefragment thereof is in a pharmaceutically acceptable excipient.

The present invention is also directed to a method of determiningwhether a compound inhibits inflammation. The method comprises combiningthe compound with (a) a cell that releases a proinflammatory cytokinewhen exposed to a vertebrate HMGB B box or biologically active fragmentthereof; and (b) the HMGB B box or biologically active fragment thereof,then determining whether the compound inhibits the release of theproinflammatory cytokine from the cell. Preferably, the HMGB B box is amammalian HMGB B box, for example, an HMGB1 B box. Preferredproinflammatory cytokines are as previously described.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of HMG1 mutants and their activityin TNF release (pg/ml).

FIG. 2A is a histogram showing the effect of 0 μg/ml, 0.01 μg/ml, 0.1μg/ml, 1 μg/ml or 10 μg/ml of B box on TNF release (pg/ml) in RAW 264.7cells.

FIG. 2B is a histogram showing the effect of 0 μg/ml, 0.01 μg/ml, 0.1μg/ml, 1 μg/ml or 10 μg/ml of B box on IL-1β release (pg/ml) in RAW264.7 cells.

FIG. 2C is a histogram showing the effect of 0 μg/ml, 0.01 μg/ml, 0.1μg/ml, 1 μg/ml or 10 μg/ml of B box on IL-6 release (pg/ml) in RAW 264.7cells.

FIG. 2D a scanned image of a blot of an RNAse protection assay, showingthe effect of B box (at 0 hours, 4 hours, 8 hours, or 24 hours afteradministration) or vector alone (at 4 hours after administration) on TNFmRNA expression in RAW 264.7 cells.

FIG. 2E is a histogram of the effect of HMG1 B box on TNF proteinrelease (pg/ml) from RAW 264.7 cells at 0 hours, 4 hours, 8 hours, 24hours, 32 hours or 48 hours after administration.

FIG. 2F is a histogram of the effect of vector on TNF protein release(pg/ml) from RAW 264.7 cells at 0 hours, 4 hours, 8 hours, 24 hours, 32hours or 48 hours after administration.

FIG. 3 is a schematic representation of HMG1 B box mutants and theiractivity in TNF release (pg/ml).

FIG. 4A is a graph of the effect of 0 μg/ml, 5 μg/ml, 10 μg/ml, or 25μg/ml of HMG1 A box protein on the release of TNF (as a percent of HMG1mediated TNF release alone) from RAW 264.7 cells.

FIG. 4B is a histogram of the effect of HMG1 (0 or 1.5 μg/ml), HMG1 Abox (0 or 10 μg/ml), or vector (0 or 10 μg/ml), alone, or in combinationon the release of TNF (as a percent of HMG1 mediated TNF release alone)from RAW 264.7 cells.

FIG. 5A is a graph of binding of ¹²⁵I-HMGB1 binding to RAW 264.7 cells(CPM/well) over time (minutes).

FIG. 5B is a histogram of the binding of ¹²⁵I-HMGB1 in the absence ofunlabeled HMGB1 or HMG1 A box for 2 hours at 4° C. (Total), or in thepresence of 5,000 molar excess of unlabeled HMG1 (HMGB1) or A box (Abox), measured as a percent of the total CPM/well.

FIG. 6 is a histogram of the effects of HMG-1 (0 μg/ml or 1 μg/ml) orHMG1 B box (0 μg/ml or 10 μg/ml), alone or in combination with anti-Bbox antibody (25 μg/ml or 100 μg/ml) or IgG (25 μg/ml or 100 μg/ml) onTNF release from RAW 264.7 cells (expressed as a percent of HMG1mediated TNF release alone).

FIG. 7A is a scanned image of a hematoxylin and eosin stained kidneysection obtained from an untreated mouse.

FIG. 7B is a scanned image of a hematoxylin and eosin stained kidneysection obtained from a mouse administered HMG1 B box.

FIG. 7C is a scanned image of a hematoxylin and eosin stained myocardiumsection obtained from an untreated mouse.

FIG. 7D is a scanned image of a hematoxylin and eosin stained myocardiumsection obtained from a mouse administered HMG1 B box.

FIG. 7E is a scanned image of a hematoxylin and eosin stained lungsection obtained from an untreated mouse.

FIG. 7F is a scanned image of a hematoxylin and eosin stained lungsection obtained from a mouse administered HMG1 B box.

FIG. 7G is a scanned image of a hematoxylin and eosin stained liversection obtained from an untreated mouse.

FIG. 7H is a scanned image of a hematoxylin and eosin stained liversection obtained from a mouse administered HMG1 B box.

FIG. 7I is a scanned image of a hematoxylin and eosin stained liversection (high magnification) obtained from an untreated mouse.

FIG. 7J is a scanned image of a hematoxylin and eosin stained liversection (high magnification) obtained from a mouse administered HMG1 Bbox.

FIG. 8 is a graph of the level of HMGB1 (ng/ml) in mice subjected tocecal ligation and puncture (CLP) over time (hours).

FIG. 9 is a graph of the effect of A Box (60 μg/mouse or 600 μg/mouse)or no treatment on survival of mice over time (days) after cecalligation and puncture (CLP).

FIG. 10A is a graph of the effect of anti-HMG1 antibody (dark circles)or no treatment (open circles) on survival of mice over time (days)after cecal ligation and puncture (CLP).

FIG. 10B is a graph of the effect of anti-HMG1 B box antiserum (▪) or notreatment (*) on the survival (days) of mice administeredlipopolysaccharide (LPS).

FIG. 11A is a histogram of the effect of anti-RAGE antibody ornon-immune IgG on TNF release from RAW 264.7 cells treated with HMG1(HMG-1), lipopolysaccharide (LPS), or HMG1 B box (B box).

FIG. 11B is a histogram of the effect of HMG1 or HMG1 B box polypeptidestimulation on activation of the NFkB-dependent ELAM promoter (measuredby luciferase activity) in RAW 264.7 cells co-transfected with a murineMyD 88-dominant negative (+MyD 88 DN) mutant (corresponding to aminoacids 146-296), or empty vector (−MyD 88 DN). Data are expressed as theratio (fold-activation) of average luciferase values from unstimulatedand stimulated cells (subtracted for background)+SD.

FIG. 11C is a histogram of the effect stimulation of CHO reporter celllines that constitutively express human TLR2 (open bars) or TLR4 (shadedbars) with IL-1, HMG1, or HMG1 B box on CD25 expression. Data areexpressed as the ratio (fold-activation) of the percent of CD25⁺ cellsin unstimulated and stimulated cell populations that were gated toexclude the lowest 5% of cells based on mean FL1 fluorescence.

FIG. 11D is a histogram of the effect of administration of anti-RAGEantibody, anti-TLR2 antibody, anti-RAGE antibody and anti-TLR2 antibodytogether, or IgG on HMG1-mediated TNF release (measured as a percent ofTNF release in the absence of antibody) in RAW 264.7 cells.

FIG. 12A is the amino acid sequence of a human HMG1 polypeptide (SEQ IDNO:1).

FIG. 12B is the amino acid sequence of rat and mouse HMG1 (SEQ ID NO:2).

FIG. 12C is the amino acid sequence of human HMG2 (SEQ ID NO:3).

FIG. 12D is the amino acid sequence of a human, mouse, and rat HMG1 Abox polypeptide (SEQ ID NO:4).

FIG. 12E is the amino acid sequence of a human, mouse, and rat HMG1 Bbox polypeptide (SEQ ID NO:5).

FIG. 12F is the nucleic acid sequence of a forward primer for human HMG1(SEQ ID NO:6).

FIG. 12G is the nucleic acid sequence of a reverse primer for human HMG1(SEQ ID NO:7).

FIG. 12H is the nucleic acid sequence of a forward primer for thecarboxy terminus mutant of human HMG1 (SEQ ID NO:8).

FIG. 12I is the nucleic acid sequence of a reverse primer for thecarboxy terminus mutant of human HMG1 (SEQ ID NO:9).

FIG. 12J is the nucleic acid sequence of a forward primer for the aminoterminus plus B box mutant of human HMG1 (SEQ ID NO:10).

FIG. 12K is the nucleic acid sequence of a reverse primer for the aminoterminus plus B box mutant of human HMG1 (SEQ ID NO:11).

FIG. 12L is the nucleic acid sequence of a forward primer for a B boxmutant of human HMG1 (SEQ ID NO:12).

FIG. 12M is the nucleic acid sequence of a reverse primer for a B boxmutant of human HMG1 (SEQ ID NO:13).

FIG. 12N is the nucleic acid sequence of a forward primer for the aminoterminus plus A box mutant of human HMG1 (SEQ ID NO:14).

FIG. 12O is the nucleic acid sequence of a reverse primer for the aminoterminus plus A box mutant of human HMG1 (SEQ ID NO:15).

FIG. 13 is a sequence alignment of HMG1 polypeptide sequence from rat(SEQ ID NO:2), mouse (SEQ ID NO:2), and human (SEQ ID NO:18).

FIG. 14A is the nucleic acid sequence of HMG1L10 (SEQ ID NO:32) encodingan HMGB polypeptide.

FIG. 14B is the polypeptide sequence of HMG1L10 (SEQ ID NO:24), an HMGBpolypeptide.

FIG. 14C is the nucleic acid sequence of HMG1L1 (SEQ ID NO:33) encodingan HMGB polypeptide.

FIG. 14D is the polypeptide sequence of HMG1L1 (SEQ ID NO:25), an HMGBpolypeptide.

FIG. 14E is the nucleic acid sequence of HMG1L4 (SEQ ID NO:34) encodingan HMGB polypeptide.

FIG. 14F is the polypeptide sequence of HMG1L4 (SEQ ID NO:26), an HMGBpolypeptide.

FIG. 14G is the nucleic acid sequence encoding the HMG polypeptidesequence of the BAC clone RP11-395A23 (SEQ ID NO:35).

FIG. 14H is the polypeptide sequence of the HMG polypeptide sequence ofthe BAC clone RP11-395A23 (SEQ ID NO:27), an HMGB polypeptide.

FIG. 14I is the nucleic acid sequence of HMG1L9 (SEQ ID NO:36) encodingan HMGB polypeptide.

FIG. 14J is the polypeptide sequence of HMG1L9 (SEQ ID NO:28), an HMGBpolypeptide.

FIG. 14K is the nucleic acid sequence of LOC122441 (SEQ ID NO:37)encoding an HMGB polypeptide.

FIG. 14L is the polypeptide sequence of LOC122441 (SEQ ID NO:29), anHMGB polypeptide.

FIG. 14M is the nucleic acid sequence of LOC139603 (SEQ ID NO:38)encoding an HMGB polypeptide.

FIG. 14N is the polypeptide sequence of LOC139603 (SEQ ID NO:30), anHMGB polypeptide.

FIG. 14O is the nucleic acid sequence of HMG1L8 (SEQ ID NO:39) encodingan HMGB polypeptide.

FIG. 14P is the polypeptide sequence of HMG1L8 (SEQ ID NO:31), an HMGBpolypeptide.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell culture, molecular biology,microbiology, cell biology, and immunology, which are well within theskill of the art. Such techniques are fully explained in the literature.See, e.g., Sambrook et al., 1989, “Molecular Cloning: A LaboratoryManual”, Cold Spring Harbor Laboratory Press; Ausubel et al. (1995),“Short Protocols in Molecular Biology”, John Wiley and Sons; Methods inEnzymology (several volumes); Methods in Cell Biology (several volumes),and Methods in Molecular Biology (several volumes).

The present invention is based on a series of discoveries that furtherelucidate various characteristics of the ability of HMGB1 to induceproduction of proinflammatory cytokines and inflammatory cytokinecascades. Specifically, it has been discovered that the proinflammatoryactive domain of HMGB1 is the B box (and in particular, the first 20amino acids of the B box), and that antibodies specific to the B boxwill inhibit proinflammatory cytokine release and inflammatory cytokinecascades, with results that can alleviate deleterious symptoms caused byinflammatory cytokine cascades. It has also been discovered that the Abox is a weak agonist of inflammatory cytokine release, andcompetitively inhibits the proinflammatory activity of the B box and ofHMGB1.

As used herein, an “HMGB polypeptide” or an “HMGB protein” is asubstantially pure, or substantially pure and isolated polypeptide thathas been separated from components that naturally accompany it, or arecombinantly produced polypeptide having the same amino acid sequence,and increases inflammation, and/or increases release of aproinflammatory cytokine from a cell, and/or increases the activity ofthe inflammatory cytokine cascade. In one embodiment, the HMGBpolypeptide has one of the above biological activities. In anotherembodiment, the HMGB polypeptide has two of the above biologicalactivities. In a third embodiment, the HMGB polypeptide has all three ofthe above biological activities.

Preferably, the HMGB polypeptide is a mammalian HMGB polypeptide, forexample, a human HMGB1 polypeptide. Preferably, the HMGB polypeptide hasat least 60%, more preferably, at least 70%, 75%, 80%, 85%, or 90%, andmost preferably at least 95% sequence identity to a sequence selectedfrom SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:18, asdetermined using the BLAST program and parameters described herein.Examples of an HMGB polypeptide include a polypeptide comprising orconsisting of the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, orSEQ ID NO:18. Preferably, the HMGB polypeptide contains a B box DNAbinding domain and/or an A box DNA binding domain, and/or an acidiccarboxyl terminus as described herein. Other examples of HMGBpolypeptides are described in GenBank Accession Numbers AAA64970,AAB08987, P07155, AAA20508, S29857, P09429, NP_(—)002119, CAA3110,S02826, U00431, X67668, NP_(—)005333, NM_(—)016957, and J04179, theentire teachings of which are incorporated herein by reference.Additional examples of HMGB polypeptides include, but are not limited tomammalian HMG1 ((HMGB1) as described, for example, in GenBank AccessionNumber U51677), HMG2 ((HMGB2) as described, for example, in GenBankAccession Number M83665), HMG-2A ((HMGB3, HMG-4) as described, forexample, in GenBank Accession Number NM_(—)005342 and NP_(—)005333),HMG14 (as described, for example, in GenBank Accession Number P05114),HMG17 (as described, for example, in GenBank Accession Number X13546),HMGI (as described, for example, in GenBank Accession Number L17131),and HMGY (as described, for example, in GenBank Accession NumberM23618); nonmammalian HMG T1 (as described, for example, in GenBankAccession Number X02666) and HMG T2 (as described, for example, inGenBank Accession Number L32859) (rainbow trout); HMG-X (as described,for example, in GenBank Accession Number D30765) (Xenopus), HMG D (asdescribed, for example, in GenBank Accession Number X71138) and HMG Z(as described, for example, in GenBank Accession Number X71139)(Drosophila); NHP10 protein (HMG protein homolog NHP 1) (as described,for example, in GenBank Accession Number Z48008) (yeast); non-histonechromosomal protein (as described, for example, in GenBank AccessionNumber O00479) (yeast); HMG1/2 like protein (as described, for example,in GenBank Accession Number Z11540) (wheat, maize, soybean); upstreambinding factor (UBF-1) (as described, for example, in GenBank AccessionNumber X53390); PMS1 protein homolog 1 (as described, for example, inGenBank Accession Number U13695); single-strand recognition protein(SSRP, structure-specific recognition protein) (as described, forexample, in GenBank Accession Number M86737); the HMG homolog TDP-1 (asdescribed, for example, in GenBank Accession Number M74017); mammaliansex-determining region Y protein (SRY, testis-determining factor) (asdescribed, for example, in GenBank Accession Number X53772); fungalproteins: mat-1 (as described, for example, in GenBank Accession NumberAB009451), ste 11 (as described, for example, in GenBank AccessionNumber X53431) and Mc 1; SOX 14 (as described, for example, in GenBankAccession Number AF107043) (as well as SOX 1 (as described, for example,in GenBank Accession Number Y13436), SOX 2 (as described, for example,in GenBank Accession Number Z31560), SOX 3 (as described, for example,in GenBank Accession Number X71135), SOX 6 (as described, for example,in GenBank Accession Number AF309034), SOX 8 (as described, for example,in GenBank Accession Number AF226675), SOX 10 (as described, forexample, in GenBank Accession Number AJ011183), SOX 12 (as described,for example, in GenBank Accession Number X73039) and SOX 21 (asdescribed, for example, in GenBank Accession Number AF107044)); lymphoidspecific factor (LEF-1) (as described, for example, in GenBank AccessionNumber X58636); T-cell specific transcription factor (TCF-1) (asdescribed, for example, in GenBank Accession Number X59869); MTT1 (asdescribed, for example, in GenBank Accession Number M62810) andSP100-HMG nuclear autoantigen (as described, for example, in GenBankAccession Number U36501).

Other examples of polypeptides having A box sequences within theminclude, but are not limited polypeptides encoded by GenBank AccessionNumbers NG_(—)00897 (HMG1L10) (and in particular by nucleotides 658-1305of NG_(—)00897, as shown in FIGS. 14A and 14B); AF076674 (HMG1L1) (andin particular by nucleotides 1-633 of AF076674, as shown in FIGS. 14Cand 14D; AF076676 (HMG1L4) (and in particular by nucleotides 1-564 ofAF076676, as shown in FIGS. 14E and 14F); AC010149 (HMG sequence fromBAC clone RP11-395A23) (and in particular by nucleotides 75503-76117 ofAC010149), as shown in FIGS. 14G and 14H); AF165168 (HMG1L9) (and inparticular by nucleotides 729-968 of AF165168, as shown in FIGS. 14I and14J); XM_(—)063129 (LOC122441) (and in particular by nucleotides 319-558of XM_(—)063129, as shown in FIGS. 14K and 14L); XM_(—)066789(LOC139603) (and in particular by nucleotides 1-258 of XM_(—)066789, asshown in FIGS. 14M and 14N); and AF165167 (HMG1L8) (and in particular bynucleotides 456-666 of AF165167, as shown in FIGS. 14O and 14LP). TheHMGB-like sequences in such polypeptides can be determined and isolatedusing methods described herein, for example, by sequence comparisons toHMGB polypeptides described herein and testing for biological activityusing method described herein or other method known in the art.

As used herein, an “HMGB A box” also referred to herein as an “A box” isa substantially pure, or substantially pure and isolated polypeptidethat has been separated from components that naturally accompany it, andconsists of an amino acid sequence that is less than a full length HMGBpolypeptide and which has one or more of the following biologicalactivities: inhibiting inflammation, and/or inhibiting release of aproinflammatory cytokine from a cell, and/or decreasing the activity ofthe inflammatory cytokine cascade. In one embodiment, the HMGB A boxpolypeptide has one of the above biological activities. In anotherembodiment, the HMGB A box polypeptide has two of the above biologicalactivities. In a third embodiment, the HMGB A box polypeptide has allthree of the above biological activities. Preferably, the HMGB A box hasno more than 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of thebiological activity of full length HMG. In one embodiment, the HMGB Abox amino acid consists of the sequence of SEQ ID NO:4, SEQ ID NO:22, orSEQ ID NO:57, or the amino acid sequence in the corresponding region ofan HMGB protein in a mammal. An HMGB A box is also a recombinantlyproduced polypeptide having the same amino acid sequence as the A boxsequences described above. Preferably, the HMGB A box is a mammalianHMGB A box, for example, a human HMG1 A box. The HMGB A box polypeptidesof the present invention preferably comprise or consist of the sequenceof SEQ ID NO:4, SEQ ID NO:22, or SEQ ID NO:57 or the amino acid sequencein the corresponding region of an HMGB protein in a mammal. An HMGB Abox often has no more than about 85 amino acids and no fewer than about4 amino acids. Examples of polypeptides having A box sequences withinthem include, but are not limited to GenBank Accession Numbers AAA64970,AAB08987, P07155, AAA20508, S29857, P09429, NP_(—)002119, CAA3110,S02826, U00431, X67668, NP_(—)005333, NM_(—)016957, and J04197; HMGBpolypeptides described herein; mammalian HMG1 ((HMGB1) as described, forexample, in GenBank Accession Number U51677), HMG2 ((HMGB2) asdescribed, for example, in GenBank Accession Number M83665), HMG-2A((HMGB3, HMG-4) as described, for example, in GenBank Accession NumbersNM_(—)005342 and NP_(—)005333), HMG14 (as described, for example, inGenBank Accession Number P05114), HMG17 (as described, for example, inGenBank Accession Number X13546), HMGI (as described, for example, inGenBank Accession Number L17131), and HMGY (as described, for example,in GenBank Accession Number M23618); nonmammalian HMG T1 (as described,for example, in GenBank Accession Number X02666) and HMG T2 (asdescribed, for example, in GenBank Accession Number L32859) (rainbowtrout); HMG-X (as described, for example, in GenBank Accession NumberD30765) (Xenopus), HMG D (as described, for example, in GenBankAccession Number X71138) and HMG Z (as described, for example, inGenBank Accession Number X71139) (Drosophila); NHP10 protein (HMGprotein homolog NHP 1) (as described, for example, in GenBank AccessionNumber Z48008) (yeast); non-histone chromosomal protein (as described,for example, in GenBank Accession Number O0479) (yeast); HMG1/2 likeprotein (as described, for example, in GenBank Accession Number Z11540)(wheat, maize, soybean); upstream binding factor (UBF-1) (as described,for example, in GenBank Accession Number X53390); PMS1 protein homolog 1(as described, for example, in GenBank Accession Number U13695);single-strand recognition protein (SSRP, structure-specific recognitionprotein) (as described, for example, in GenBank Accession NumberM86737); the HMG homolog TDP-1 (as described, for example, in GenBankAccession Number M74017); mammalian sex-determining region Y protein(SRY, testis-determining factor) (as described, for example, in GenBankAccession Number X53772); fungal proteins: mat-1 (as described, forexample, in GenBank Accession Number AB009451), ste 11 (as described,for example, in GenBank Accession Number x53431) and Mc 1; SOX 14 (asdescribed, for example, in GenBank Accession Number AF107043) (as wellas SOX 1 (as described, for example, in GenBank Accession NumberY13436), SOX 2 (as described, for example, in GenBank Accession NumberZ31560), SOX 3 (as described, for example, in GenBank Accession NumberX71135), SOX 6 (as described, for example, in GenBank Accession NumberAF309034), SOX 8 (as described, for example, in GenBank Accession NumberAF226675), SOX 10 (as described, for example, in GenBank AccessionNumber AJ001183), SOX 12 (as described, for example, in GenBankAccession Number X73039) and SOX 21 (as described, for example, inGenBank Accession Number AF107044)); lymphoid specific factor (LEF-1)(as described, for example, in GenBank Accession Number X58636); T-cellspecific transcription factor (TCF-1) (as described, for example, inGenBank Accession Number X59869); MTT1 (as described, for example, inGenBank Accession Number M62810) and SP100-HMG nuclear autoantigen (asdescribed, for example, in GenBank Accession Number U36501).

Other examples of polypeptides having A box sequences within theminclude, but are not limited polypeptides encoded by GenBank AccessionNumbers NG_(—)00897 (HMG1L10) (and in particular by nucleotides 658-1305of NG_(—)00897, as shown in FIGS. 14A and 14B); AF076674 (HMG1L1) (andin particular by nucleotides 1-633 of AF076674, as shown in FIGS. 14Cand 14D; AF076676 (HMG1L4) (and in particular by nucleotides 1-564 ofAF076676, as shown in FIGS. 14E and 14F); AC010149 (HMG sequence fromBAC clone RP11-395A23) (and in particular by nucleotides 75503-76117 ofAC010149), as shown in FIGS. 14G and 14H); AF165168 (HMG1L9) (and inparticular by nucleotides 729-968 of AF165168, as shown in FIGS. 141 and14J); XM_(—)063129 (LOC122441) (and in particular by nucleotides 319-558of XM_(—)063129, as shown in FIGS. 14K and 14L); XM_(—)066789(LOC139603) (and in particular by nucleotides 1-258 of XM_(—)066789, asshown in FIGS. 14M and 14N); and AF165167 (HMG1L8) (and in particular bynucleotides 456-666 of AF165167, as shown in FIGS. 14O and 14LP). The Abox sequences in such polypeptides can be determined and isolated usingmethods described herein, for example, by sequence comparisons to Aboxes described herein and testing for biological activity using methoddescribed herein or other method known in the art.

Examples of HMGB A box polypeptide sequences include the followingsequences: PDASVNFSEF SKKCSERWKT MSAKEKGKFE DMAKADKARY EREMKTYIPP KGET(Human HMGB1; SEQ ID NO: 4); DSSVNFAEF SKKCSERWKT MSAKEKSKFE DMAKSDKARYDREMKNYVPP KGDK; (Human HMGB2; SEQ ID NO: 41); PEVPVNFAEF SKKCSERWKTVSGKEKSKFD EMAKADKVRY DREMKDYGPA KGGK (Human HMGB3; SEQ ID NO: 42);PDASVNFSEF SKKCSERWKT MSAKEKGKFE DMAKADKARY EREMKTYIPP KGET (HMG1L10;SEQ ID NO: 43); SDASVNFSEF SNKCSERWKT MSAKEKGKFE DMAKADKTHY ERQMKTYIPPKGET (HMG1L1; SEQ ID NO: 44); PDASVNFSEF SKKCSERWKA MSAKDKGKFEDMAKVDKADY EREMKTYIPP KGET (HMG1L4; SEQ ID NO: 45); PDASVKFSEFLKKCSETWKT IFAKEKGKFE DMAKADKAHY EREMKTYIPP KGEK (HMG sequence from BACclone RP11-395A23; SEQ ID NO: 46); PDASINFSEF SQKCPETWKT TIAKEKGKFEDMAKADKAHY EREMKTYIPP KGET (HMG1L9; SEQ ID NO: 47); PDASVNSSEFSKKCSERWKTMPTKQGKFE DMAKADRAH (HMG1L8; SEQ ID NO: 48); PDASVNFSEFSKKCLVRGKT MSAKEKGQFE AMARADKARY EREMKTYIP PKGET (LOC122441; SEQ ID NO:49); LDASVSFSEF SNKCSERWKT MSVKEKGKFE DMAKADKACY EREMKIYPYL KGRQ(LOC139603; SEQ ID NO: 50); and GKGDPKKPRG KMSSYAFFVQ TCREEHKKKHPDASVNFSEF SKKCSERWKT MSAKEKGKFE DMAKADKARY EREMKTYIPP KGET (human HMGB1A box; SEQ ID NO: 57).

The present invention also features non-naturally occurring HMGB Aboxes. Preferably, a non-naturally occurring HMGB A box has at least60%, more preferably, at least 70%, 75%, 80%, 85%, or 90%, and mostpreferably at least 95% sequence identity to the sequence of SEQ IDNO:4, SEQ ID NO:22, or SEQ ID NO:57, as determined using the BLASTprogram and parameters described herein and one of more of thebiological activities of an HMGB A box.

The present invention also features A box biologically active fragments.By an “A box fragment that has A box biological activity” or an “A boxbiologically active fragment” is meant a fragment of an HMGB A box thathas the activity of an HMGB A box, as described herein. For example, theA box fragment can decrease release of a pro-inflammatory cytokine froma vertebrate cell, decrease inflammation, and/or decrease activity ofthe inflammatory cytokine cascade. A box fragments can be generatedusing standard molecular biology techniques and assaying the function ofthe fragment by determining if the fragment, when administered to a cellinhibits release of a proinflammatory cytokine from the cell, forexample using methods described herein. A box biologically activefragments can be used in the methods described herein in which fulllength A box polypeptides are used, for example, inhibiting release of aproinflammatory cytokine from a cell, or treating a patient having acondition characterized by activation of an inflammatory cytokinecascade.

As used herein, an “HMGB B box” also referred to herein as a “B box” isa substantially pure, or substantially pure and isolated polypeptidethat has been separated from components that naturally accompany it, andconsists of an amino acid sequence that is less than a full length HMGBpolypeptide and has one or more of the following biological activities:increasing inflammation, increasing release of a proinflammatorycytokine from a cell, and or increasing the activity of the inflammatorycytokine cascade. In one embodiment, the HMGB B box polypeptide has oneof the above biological activities. In another embodiment, the HMGB Bbox polypeptide has two of the above biological activities. In a thirdembodiment, the HMGB B box polypeptide has all three of the abovebiological activities. Preferably, the HMGB B box has at least 25%, 30%,40%, 50%, 60%, 70%, 80% or 90% of the biological activity of full lengthHMG. In another embodiment, the HMGB B box does not comprise an HMGB Abox. In another embodiment, the HMGB B box is a polypeptide that isabout 90%, 80%, 70%, 60%, 50%, 40%, 35%, 30%, 25%, or 20% the length ofa full length HMGB1 polypeptide. In another embodiment, the HMGB boxcomprises or consists of sequence of SEQ ID NO:5, SEQ ID NO:20, or SEQID NO:58, or the amino acid sequence in the corresponding region of anHMGB protein in a mammal, but is still less than the full length HMGBpolypeptide. An HMGB B box polypeptide is also a recombinantly producedpolypeptide having the same amino acid sequence as an HMGB B boxpolypeptide described above. Preferably, the HMGB B box is a mammalianHMGB B box, for example, a human HMGB1 B box. An HMGB B box often has nomore than about 85 amino acids and no fewer than about 4 amino acids.Examples of polypeptides having B box sequences within them include, butare not limited to GenBank Accession Numbers AAA64970, AAB08987, P07155,AAA20508, S29857, P09429, NP_(—)002119, CAA31110, S02826, U00431,X67668, NP_(—)005333, NM 016957, and J04197; HMG polypeptides describedherein; mammalian HMG1 ((HMGB1) as described, for example, in GenBankAccession Number U51677), HMG2 ((HMGB2) as described, for example, inGenBank Accession Number M83665), HMG-2A ((HMGB3, HMG-4) as described,for example, in GenBank Accession Numbers NM_(—)005342 andNP_(—)005333), HMG14 (as described, for example, in GenBank AccessionNumber P05114), HMG17 (as described, for example, in GenBank AccessionNumber X13546), HMGI (as described, for example, in GenBank AccessionNumber L17131), and HMGY (as described, for example, in GenBankAccession Number M23618); nonmammalian HMG T1 (as described, forexample, in GenBank Accession Number X02666) and HMG T2 (as described,for example, in GenBank Accession Number L32859) (rainbow trout); HMG-X(as described, for example, in GenBank Accession Number D30765)(Xenopus), HMG D (as described, for example, in GenBank Accession NumberX71138) and HMG Z (as described, for example, in GenBank AccessionNumber X71139) (Drosophila); NHP10 protein (HMG protein homolog NHP 1)(as described, for example, in GenBank Accession Number Z48008) (yeast);non-histone chromosomal protein (as described, for example, in GenBankAccession Number O00479) (yeast); HMG1/2 like protein (as described, forexample, in GenBank Accession Number Z11540) (wheat, maize, soybean);upstream binding factor (UBF-1) (as described, for example, in GenBankAccession Number X53390); PMS1 protein homolog 1 (as described, forexample, in GenBank Accession Number U13695); single-strand recognitionprotein (SSRP, structure-specific recognition protein) (as described,for example, in GenBank Accession Number M86737); the HMG homolog TDP-1(as described, for example, in GenBank Accession Number M74017);mammalian sex-determining region Y protein (SRY, testis-determiningfactor) (as described, for example, in GenBank Accession Number X53772);fungal proteins: mat-1 (as described, for example, in GenBank AccessionNumber AB009451), ste 11 (as described, for example, in GenBankAccession Number x53431) and Mc 1; SOX 14 (as described, for example, inGenBank Accession Number AF107043) (as well as SOX 1 (as described, forexample, in GenBank Accession Number Y13436), SOX 2 (as described, forexample, in GenBank Accession Number Z31560), SOX 3 (as described, forexample, in GenBank Accession Number X71135), SOX 6 (as described, forexample, in GenBank Accession Number AF309034), SOX 8 (as described, forexample, in GenBank Accession Number AF226675), SOX 10 (as described,for example, in GenBank Accession Number AJ011183), SOX 12 (asdescribed, for example, in GenBank Accession Number X73039) and SOX 21(as described, for example, in GenBank Accession Number AF107044));lymphoid specific factor (LEF-1) (as described, for example, in GenBankAccession Number X58636); T-cell specific transcription factor (TCF-1)(as described, for example, in GenBank Accession Number X59869); MTT1(as described, for example, in GenBank Accession Number M62810) andSP100-HMG nuclear autoantigen (as described, for example, in GenBankAccession Number U36501).

Other examples of polypeptides having B box sequences within theminclude, but are not limited polypeptides encoded by GenBank AccessionNumbers NG_(—)00897 (HMG1L10) (and in particular by nucleotides 658-1305of NG_(—)00897, as shown in FIGS. 14A and 14B); AF076674 (HMG1L1) (andin particular by nucleotides 1-633 of AF076674, as shown in FIGS. 14Cand 14D; AF076676 (HMG1L4) (and in particular by nucleotides 1-564 ofAF076676, as shown in FIGS. 14E and 14F); and AC010149 (HMG sequencefrom BAC clone RP11-395A23) (and in particular by nucleotides75503-76117 of AC010149), as shown in FIGS. 14G and 14H). The B boxsequences in such polypeptides can be determined and isolated usingmethods described herein, for example, by sequence comparisons to Bboxes described herein and testing for biological activity using methoddescribed herein or other method known in the art.

Examples of HMGB B box polypeptide sequences include the followingsequences: FKDPNAPKRP PSAFFLFCSE YRPKIKGEHP GLSIGDVAKK LGEMWNNTAADDKQPYEKKA AKLKEKYEKD IAAY (human HMGB1; SEQ ID NO: 51); KKDPNAPKRPPSAFFLFCSE HRPKIKSEHP GLSIGDTAKK LGEMWSEQSA KDKQPYEQKA AKLKEKYEKD IAAY(human HMGB2; SEQ ID NO: 52); FKDPNAPKRL PSAFFLFCSE YRPKIKGEHPGLSIGDVAKK LGEMWNNTAA DDKQPYEKKA AKLKEKYEKD IAAY (HMG1L10; SEQ ID NO:53); FKDPNAPKRP PSAFFLFCSE YHPKIKGEHP GLSIGDVAKK LGEMWNNTAA DDKQPGEKKAAKLKEKYEKD IAAY (HMG1L1; SEQ ID NO: 54); FKDSNAPKRP PSAFLLFCSEYCPKIKGEHP GLPISDVAKK LVEMWNNTFA DDKQLCEKKA AKLKEKYKKD TATY (HMG1L4; SEQID NO: 55); FKDPNAPKRP PSAFFLFCSE YRPKIKGEHP GLSIGDVVKK LAGMWNNTAAADKQFYEKKA AKLKEKYKKD IAAY (HMG sequence from BAC clone RP11-359A23; SEQID NO: 56); and FKDPNAPKRP PSAFFLFCSE YRPKIKGEHP GLSIGDVAKK LGEMWNNTAADDKQPYEKKA AKLKEKYEKD IAAYRAKGKP DAAKKGVVKA EK (human HMGB1 box; SEQ IDNO: 58).

The present invention also includes non-naturally occurring HMGB B boxpolypeptides. Preferably, a non-naturally occurring HMGB B boxpolypeptide has at least 60%, more preferably, at least 70%, 75%, 80%,85%, or 90%, and most preferably at least 95% sequence identity to thesequence of SEQ ID NO:5, SEQ ID NO:20, or SEQ ID NO:58, as determinedusing the BLAST program and parameters described herein. Preferably, theHMGB B box consists of the sequence of SEQ ID NO:5, SEQ ID NO:20, or SEQID NO:58, or the amino acid sequence in the corresponding region of anHMGB protein in a mammal.

In other embodiments, the present invention is directed to a polypeptidecomprising a vertebrate HMGB B box or a fragment thereof that has B boxbiological activity, or a non-naturally occurring HMGB B box but notcomprising a full length HMG. By a “B Box fragment that has B boxbiological activity” or a “B box biologically active fragment” is meanta fragment of an HMGB B box that has the activity of an HMGB B box. Forexample, the B box fragment can induce release of a pro-inflammatorycytokine from a vertebrate cell or increase inflammation, or induce theinflammatory cytokine cascade. An example of such a B box fragment isthe fragment comprising the first 20 amino acids of the HMGB1 B box (SEQID NO:16 or SEQ ID NO:23), as described herein. B box fragments can begenerated using standard molecular biology techniques and assaying thefunction of the fragment by determining if the fragment, whenadministered to a cell increase release of a proinflammatory cytokinefrom the cell, compared to a suitable control, for example, usingmethods described herein.

As used herein, a “cytokine” is a soluble protein or peptide which isnaturally produced by mammalian cells and which acts in vivo as ahumoral regulator at micro- to picomolar concentrations. Cytokines can,either under normal or pathological conditions, modulate the functionalactivities of individual cells and tissues. A proinflammatory cytokineis a cytokine that is capable of causing any of the followingphysiological reactions associated with inflammation: vasodilation,hyperemia, increased permeability of vessels with associated edema,accumulation of granulocytes and mononuclear phagocytes, or depositionof fibrin. In some cases, the proinflammatory cytokine can also causeapoptosis, such as in chronic heart failure, where TNF has been shown tostimulate cardiomyocyte apoptosis (Pulkki, Ann. Med. 29: 339-343, 1997;and Tsutsui et al., Immunol. Rev. 174:192-209, 2000).

Nonlimiting examples of proinflammatory cytokines are tumor necrosisfactor (TNF), interleukin (IL)-1α, IL-1β, IL-6, IL-8, IL-18, interferonγ, HMG-1, platelet-activating factor (PAF), and macrophage migrationinhibitory factor (MIF).

Proinflammatory cytokines are to be distinguished from anti-inflammatorycytokines, such as IL-4, IL-10, and IL-13, which are not mediators ofinflammation.

In many instances, proinflammatory cytokines are produced in aninflammatory cytokine cascade, defined herein as an in vivo release ofat least one proinflammatory cytokine in a mammal, wherein the cytokinerelease affects a physiological condition of the mammal. Thus, aninflammatory cytokine cascade is inhibited in embodiments of theinvention where proinflammatory cytokine release causes a deleteriousphysiological condition.

HMGB A boxes and HMGB B boxes, either naturally occurring ornon-naturally occurring, include polypeptides that have sequenceidentity to the HMGB A boxes and HMGB B boxes described above. As usedherein, two polypeptides (or a region of the polypeptides) aresubstantially homologous or identical when the amino acid sequences areat least about 60%, 70%, 75%, 80%, 85%, 90% or 95% or more homologous oridentical. The percent identity of two amino acid sequences (or twonucleic acid sequences) can be determined by aligning the sequences foroptimal comparison purposes (e.g., gaps can be introduced in thesequence of a first sequence). The amino acids or nucleotides atcorresponding positions are then compared, and the percent identitybetween the two sequences is a function of the number of identicalpositions shared by the sequences (i.e., % identity=# of identicalpositions/total # of positions×100). In certain embodiments, the lengthof the HMGB polypeptide, HMGB A box polypeptide, or HMGB B boxpolypeptide aligned for comparison purposes is at least 30%, preferably,at least 40%, more preferably, at least 60%, and even more preferably,at least 70%, 80%, 90%, or 100% of the length of the reference sequence,for example, those sequence provided in FIGS. 12A-12E, and SEQ ID NOS:18, 20, and 22. The actual comparison of the two sequences can beaccomplished by well-known methods, for example, using a mathematicalalgorithm. A preferred, non-limiting example of such a mathematicalalgorithm is described in Karlin et al. (Proc. Natl. Acad. Sci. USA,90:5873-5877, 1993). Such an algorithm is incorporated into the BLASTNand BLASTX programs (version 2.2) as described in Schaffer et al.(Nucleic Acids Res., 29:2994-3005, 2001). When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., BLASTN) can be used. See the Internet site for the NationalCenter for Biotechnology Information (NCBI). In one embodiment, thedatabase searched is a non-redundant (NR) database, and parameters forsequence comparison can be set at: no filters; Expect value of 10; WordSize of 3; the Matrix is BLOSUM62; and Gap Costs have an Existence of 11and an Extension of 1.

Another preferred, non-limiting example of a mathematical algorithmutilized for the comparison of sequences is the algorithm of Myers andMiller, CABIOS (1989). Such an algorithm is incorporated into the ALIGNprogram (version 2.0), which is part of the GCG (Accelrys) sequencealignment software package. When utilizing the ALIGN program forcomparing amino acid sequences, a PAM120 weight residue table, a gaplength penalty of 12, and a gap penalty of 4 can be used. Additionalalgorithms for sequence analysis are known in the art and includeADVANCE and ADAM as described in Torellis and Robotti (Comput. Appl.Biosci., 10: 3-5, 1994); and FASTA described in Pearson and Lipman(Proc. Natl. Acad. Sci USA, 85: 2444-2448, 1988).

In another embodiment, the percent identity between two amino acidsequences can be accomplished using the GAP program in the GCG softwarepackage (Accelrys, San Diego, Calif.) using either a Blossom 63 matrixor a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and a lengthweight of 2, 3, or 4. In yet another embodiment, the percent identitybetween two nucleic acid sequences can be accomplished using the GAPprogram in the GCG software package (Accelrys, San Diego, Calif.), usinga gap weight of 50 and a length weight of 3.

A Box Polypeptides and Biologically Active Fragments Thereof

As described above, the present invention is directed to a polypeptidecomposition comprising a vertebrate HMGB A box, or a biologically activefragment thereof which can inhibit release of a proinflammatory cytokinefrom a vertebrate cell treated with HMG, or which can be used to treat acondition characterized by activation of an inflammatory cytokinecascade.

When referring to the effect of any of the compositions or methods ofthe invention on the release of proinflammatory cytokines, the use ofthe terms “inhibit” or “decrease” encompasses at least a small butmeasurable reduction in proinflammatory cytokine release. In preferredembodiments, the release of the proinflammatory cytokine is inhibited byat least 20% over non-treated controls; in more preferred embodiments,the inhibition is at least 50%; in still more preferred embodiments, theinhibition is at least 70%, and in the most preferred embodiments, theinhibition is at least 80%. Such reductions in proinflammatory cytokinerelease are capable of reducing the deleterious effects of aninflammatory cytokine cascade in in vivo embodiments.

Because all vertebrate HMGB A boxes show a high degree of sequenceconservation (see, for example, FIG. 13 for an amino acids sequencecomparison of rat, mouse, and human HMGB polypeptides), it is believedthat any vertebrate HMGB A box can inhibit release of a proinflammatorycytokine from a vertebrate cell treated with HMG. Therefore, anyvertebrate HMGB A box is within the scope of the invention. Preferably,the HMGB A box is a mammalian HMGB A box, for example, a mammalian HMGB1A box, such as a human HMGB1 A box provided herein as SEQ ID NO:4, SEQID NO:22, or SEQ ID NO:57. Also included in the present invention arefragments of the HMGB1 A box having HMGB A box biological activity, asdescribed herein.

It would also be recognized by the skilled artisan that non-naturallyoccurring HMGB A boxes (or biologically active fragments thereof) can becreated without undue experimentation, which would inhibit release of aproinflammatory cytokine from a vertebrate cell treated with avertebrate HMG. These non-naturally occurring functional A boxes can becreated by aligning amino acid sequences of HMGB A boxes from differentsources, and making one or more substitutions in one of the sequences atamino acid positions where the A boxes differ. The substitutions arepreferably made using the same amino acid residue that occurs in thecompared A box. Alternatively, a conservative substitution is made fromeither of the residues.

Conservative amino acid substitutions refer to the interchangeability ofresidues having similar side chains. Conservatively substituted aminoacids can be grouped according to the chemical properties of their sidechains. For example, one grouping of amino acids includes those aminoacids have neutral and hydrophobic side chains (a, v, l, i, p, w, f, andm); another grouping is those amino acids having neutral and polar sidechains (g, s, t, y, c, n, and q); another grouping is those amino acidshaving basic side chains (k, r, and h); another grouping is those aminoacids having acidic side chains (d and e); another grouping is thoseamino acids having aliphatic side chains (g, a, v, l, and i); anothergrouping is those amino acids having aliphatic-hydroxyl side chains (sand t); another grouping is those amino acids having amine-containingside chains (m, q, k, r, and h); another grouping is those amino acidshaving aromatic side chains (f, y, and w); and another grouping is thoseamino acids having sulfur-containing side chains c and m). Preferredconservative amino acid substitutions groups are: r-k; e-d, y-f, l-m;v-i, and q-h.

While a conservative amino acid substitution would be expected topreserve the biological activity of an HMGB A box polypeptide, thefollowing is one example of how non-naturally occurring A boxpolypeptides can be made by comparing the human HMGB1 A box (SEQ IDNO:4) with residues 32 to 85 of SEQ ID NO:3 of the human HMG2 A box (SEQID NO:17).

HMGB1 pdasvnfsef skkcserwkt msakekgkfe dmakadkary eremktyipp kget

HMGB2 pdssvnfaef skkcserwkt msakekskfe dmaksdkary dremknyvpp kgdk

A non-naturally occurring HMGB A box can be created by, for example, bysubstituting the alanine (a) residue at the third position in the HMGB1A box with the serine (s) residue that occurs at the third position ofthe HMGB2 A box. The skilled artisan would know that the substitutionwould provide a functional non-naturally occurring A box because the sresidue functions at that position in the HMGB2 A box. Alternatively,the third position of the HMGB1 A box can be substituted with any aminoacid that is conservative to alanine or serine, such as glycine (g),threonine (t), valine (v) or leucine (1). The skilled artisan wouldrecognize that these conservative substitutions would be expected toresult in a functional A box because A boxes are not invariant at thethird position, so a conservative substitution would provide an adequatestructural substitute for an amino acid that is naturally occurring atthat position.

Following the above method, a great many non-naturally occurring HMGB Aboxes could be created without undue experimentation which would beexpected to be functional, and the functionality of any particularnon-naturally occurring HMGB A box could be predicted with adequateaccuracy. In any event, the functionality of any non-naturally occurringHMGB A box could be determined without undue experimentation by simplyadding it to cells along with an HMG, and determine whether the A boxinhibits release of a proinflammatory cytokine by the cells, using, forexample, methods described herein.

The cell from which the A box or an A box biologically active fragmentwill inhibit the release of HMG-induced proinflammatory cytokines can beany cell that can be induced to produce a proinflammatory cytokine. Inpreferred embodiments, the cell is an immune cell, for example, amacrophage, a monocyte, or a neutrophil. In the most preferredembodiment, the cell is a macrophage.

Polypeptides comprising an A box or A box biologically active fragmentthat can inhibit the production of any single proinflammatory cytokine,now known or later discovered, are within the scope of the presentinvention. Preferably, the antibodies can inhibit the production of TNF,IL-1β, or IL-6. Most preferably, the antibodies can inhibit theproduction of any proinflammatory cytokines produced by the vertebratecell.

The present invention is also directed to a composition comprising anyof the above-described polypeptides, in a pharmaceutically acceptableexcipient. In these embodiments, the composition can inhibit a conditioncharacterized by activation of an inflammatory cytokine cascade. Thecondition can be one where the inflammatory cytokine cascade causes asystemic reaction, such as with endotoxic shock. Alternatively, thecondition can be mediated by a localized inflammatory cytokine cascade,as in rheumatoid arthritis. Nonlimiting examples of conditions which canbe usefully treated using the present invention include those conditionsenumerated in the background section of this specification. Preferably,the condition is appendicitis, peptic, gastric or duodenal ulcers,peritonitis, pancreatitis, ulcerative, pseudomembranous, acute orischemic colitis, diverticulitis, epiglottitis, achalasia, cholangitis,cholecystitis, hepatitis, Crohn's disease, enteritis, Whipple's disease,asthma, allergy, anaphylactic shock, immune complex disease, organischemia, reperfusion injury, organ necrosis, hay fever, sepsis,septicemia, endotoxic shock, cachexia, hyperpyrexia, eosinophilicgranuloma, granulomatosis, sarcoidosis, septic abortion, epididymitis,vaginitis, prostatitis, urethritis, bronchitis, emphysema, rhinitis,cystic fibrosis, pneumonitis,pneumoultramicroscopicsilicovolcanoconiosis, alvealitis, bronchiolitis,pharyngitis, pleurisy, sinusitis, influenza, respiratory syncytial virusinfection, herpes infection, HIV infection, hepatitis B virus infection,hepatitis C virus infection, disseminated bacteremia, Dengue fever,candidiasis, malaria, filariasis, amebiasis, hydatid cysts, burns,dermatitis, dermatomyositis, sunburn, urticaria, warts, wheals,vasulitis, angiitis, endocarditis, arteritis, atherosclerosis,thrombophlebitis, pericarditis, myocarditis, myocardial ischemia,periarteritis nodosa, rheumatic fever, Alzheimer's disease, coeliacdisease, congestive heart failure, adult respiratory distress syndrome,meningitis, encephalitis, multiple sclerosis, cerebral infarction,cerebral embolism, Guillame-Barre syndrome, neuritis, neuralgia, spinalcord injury, paralysis, uveitis, arthritides, arthralgias,osteomyelitis, fasciitis, Paget's disease, gout, periodontal disease,rheumatoid arthritis, synovitis, myasthenia gravis, thryoiditis,systemic lupus erythematosus, Goodpasture's syndrome, Behcets'ssyndrome, allograft rejection, graft-versus-host disease, Type Idiabetes, ankylosing spondylitis, Berger's disease, Type I diabetes,ankylosing spondylitis, Retier's syndrome, or Hodgkins disease. In morepreferred embodiments, the condition is appendicitis, peptic, gastric orduodenal ulcers, peritonitis, pancreatitis, ulcerative,pseudomembranous, acute or ischemic colitis, hepatitis, Crohn's disease,asthma, allergy, anaphylactic shock, organ ischemia, reperfusion injury,organ necrosis, hay fever, sepsis, septicemia, endotoxic shock,cachexia, septic abortion, disseminated bacteremia, burns, Alzheimer'sdisease, coeliac disease, congestive heart failure, adult respiratorydistress syndrome, cerebral infarction, cerebral embolism, spinal cordinjury, paralysis, allograft rejection or graft-versus-host disease. Inthe most preferred embodiments, the condition is endotoxic shock orallograft rejection. Where the condition is allograft rejection, thecomposition may advantageously also include an immunosuppressant that isused to inhibit allograft rejection, such as cyclosporin.

The excipient included with the polypeptide in these compositions ischosen based on the expected route of administration of the compositionin therapeutic applications. The route of administration of thecomposition depends on the condition to be treated. For example,intravenous injection may be preferred for treatment of a systemicdisorder such as endotoxic shock, and oral administration may bepreferred to treat a gastrointestinal disorder such as a gastric ulcer.The route of administration and the dosage of the composition to beadministered can be determined by the skilled artisan without undueexperimentation in conjunction with standard dose-response studies.Relevant circumstances to be considered in making those determinationsinclude the condition or conditions to be treated, the choice ofcomposition to be administered, the age, weight, and response of theindividual patient, and the severity of the patient's symptoms. Thus,depending on the condition, the antibody composition can be administeredorally, parenterally, intranasally, vaginally, rectally, lingually,sublingually, bucally, intrabuccaly and transdermally to the patient.

Accordingly, compositions designed for oral, lingual, sublingual, buccaland intrabuccal administration can be made without undue experimentationby means well known in the art, for example, with an inert diluent orwith an edible carrier. The compositions may be enclosed in gelatincapsules or compressed into tablets. For the purpose of oral therapeuticadministration, the pharmaceutical compositions of the present inventionmay be incorporated with excipients and used in the form of tablets,troches, capsules, elixirs, suspensions, syrups, wafers, chewing gumsand the like.

Tablets, pills, capsules, troches and the like may also contain binders,recipients, disintegrating agent, lubricants, sweetening agents, andflavoring agents. Some examples of binders include microcrystallinecellulose, gum tragacanth or gelatin. Examples of excipients includestarch or lactose. Some examples of disintegrating agents includealginic acid, corn starch and the like. Examples of lubricants includemagnesium stearate or potassium stearate. An example of a glidant iscolloidal silicon dioxide. Some examples of sweetening agents includesucrose, saccharin and the like. Examples of flavoring agents includepeppermint, methyl salicylate, orange flavoring and the like. Materialsused in preparing these various compositions should be pharmaceuticallypure and non-toxic in the amounts used.

The compositions of the present invention can easily be administeredparenterally such as, for example, by intravenous, intramuscular,intrathecal or subcutaneous injection. Parenteral administration can beaccomplished by incorporating the antibody compositions of the presentinvention into a solution or suspension. Such solutions or suspensionsmay also include sterile diluents such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents. Parenteral formulations may also includeantibacterial agents such as, for example, benzyl alcohol or methylparabens, antioxidants such as, for example, ascorbic acid or sodiumbisulfite and chelating agents such as EDTA. Buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose may also be added. The parenteralpreparation can be enclosed in ampules, disposable syringes or multipledose vials made of glass or plastic.

Rectal administration includes administering the pharmaceuticalcompositions into the rectum or large intestine. This can beaccomplished using suppositories or enemas. Suppository formulations caneasily be made by methods known in the art. For example, suppositoryformulations can be prepared by heating glycerin to about 120° C.,dissolving the antibody composition in the glycerin, mixing the heatedglycerin after which purified water may be added, and pouring the hotmixture into a suppository mold.

Transdermal administration includes percutaneous absorption of thecomposition through the skin. Transdermal formulations include patches,ointments, creams, gels, salves and the like.

The present invention includes nasally administering to the mammal atherapeutically effective amount of the composition. As used herein,nasally administering or nasal administration includes administering thecomposition to the mucous membranes of the nasal passage or nasal cavityof the patient. As used herein, pharmaceutical compositions for nasaladministration of a composition include therapeutically effectiveamounts of the agonist prepared by well-known methods to beadministered, for example, as a nasal spray, nasal drop, suspension,gel, ointment, cream or powder. Administration of the composition mayalso take place using a nasal tampon or nasal sponge.

The polypeptide compositions described herein can also include anantagonist of an early sepsis mediator. As used herein, an early sepsismediator is a proinflammatory cytokine that is released from cells soon(i.e., within 30-60 min.) after induction of an inflammatory cytokinecascade (e.g., exposure to LPS). Nonlimiting examples of these cytokinesare TNF, IL-1α, IL-1β, IL-6, PAF, and MIF. Also included as early sepsismediators are receptors for these cytokines (for example, tumor necrosisfactor receptor type 1) and enzymes required for production of thesecytokines, for example, interleukin-1β converting enzyme). Antagonistsof any early sepsis mediator, now known or later discovered, can beuseful for these embodiments by further inhibiting an inflammatorycytokine cascade.

Nonlimiting examples of antagonists of early sepsis mediators areantisense compounds that bind to the mRNA of the early sepsis mediator,preventing its expression (see, e.g., Ojwang et al., Biochemistry36:6033-6045, 1997; Pampfer et al., Biol. Reprod. 52:1316-1326, 1995;U.S. Pat. No. 6,228,642; Yahata et al., Antisense Nucleic Acid Drug Dev.6:55-61, 1996; and Taylor et al., Antisense Nucleic Acid Drug Dev.8:199-205, 1998), ribozymes that specifically cleave the mRNA of theearly sepsis mediator (see, e.g., Leavitt et al., Antisense Nucleic AcidDrug Dev. 10: 409-414, 2000; Kisich et al., 1999; and Hendrix et al.,Biochem. J. 314 (Pt. 2): 655-661, 1996), and antibodies that bind to theearly sepsis mediator and inhibit their action (see, e.g., Kam andTargan, Expert Opin. Pharmacother. 1: 615-622, 2000; Nagahira et al., J.Immunol. Methods 222, 83-92, 1999; Lavine et al., J. Cereb. Blood FlowMetab. 18: 52-58, 1998; and Holmes et al., Hybridoma 19: 363-367, 2000).Any antagonist of an early sepsis mediator, now known or laterdiscovered, is envisioned as within the scope of the invention. Theskilled artisan can determine the amount of early sepsis mediator to usein these compositions for inhibiting any particular inflammatorycytokine cascade without undue experimentation with routinedose-response studies.

B Box Polypeptides, Biologically Active Fragments Thereof, andAntibodies Thereto

As described above, the present invention is directed to a polypeptidecomposition comprising a vertebrate HMGB B box, or a biologically activefragment thereof which can increase release of a proinflammatorycytokine from a vertebrate cell treated with HMG.

When referring to the effect of any of the compositions or methods ofthe invention on the release of proinflammatory cytokines, the use ofthe term “increase” encompasses at least a small but measurable rise inproinflammatory cytokine release. In preferred embodiments, the releaseof the proinflammatory cytokine is increased by at least 1.5-fold, atleast 2-fold, at least 5-fold, or at least 10-fold over non-treatedcontrols. Such increases in proinflammatory cytokine release are capableof increasing the effects of an inflammatory cytokine cascade in in vivoembodiments. Such polypeptides can also be used to induce weight lossand/or treat obesity.

Because all HMGB B boxes show a high degree of sequence conservation(see, for example, FIG. 13 for an amino acids sequence comparison ofrat, mouse, and human HMGB polypeptides), it is believed that functionalnon-naturally occurring HMGB B boxes can be created without undueexperimentation by making one or more conservative amino acidsubstitutions, or by comparing naturally occurring vertebrate B boxesfrom different sources and substituting analogous amino acids, as wasdiscussed above with respect to the creation of functional non-naturallyoccurring A boxes. In particularly preferred embodiments, the B boxcomprises SEQ ID NO:5, SEQ ID NO: 20, or SEQ ID NO:58, which are thesequences (three different lengths) of the human HMGB1 B box, or is afragment of an HMGB B box that has B box biological activity. Forexample, a 20 amino acid sequence contained within SEQ ID NO:20contributes to the function of the B box. This 20 amino acid B-boxfragment has the following amino acid sequence: fkdpnapkrl psafflfcse(SEQ ID NO:23). Another example of an HMGB B box biologically activefragment consists of amino acids 1-20 of SEQ ID NO:5 (napkrppsafflfcseyrpk; SEQ ID NO:16).

The invention is also directed to a purified preparation of antibodiesthat specifically bind to a vertebrate high mobility group protein (HMG)B box, but do not specifically bind to non-B box epitopes of HMGB1. Inthese embodiments, the antibodies can inhibit a biological activity of aB box polypeptide, for example, the release of a proinflammatorycytokine from a vertebrate cell induced by HMG.

To make antibodies specific to the HMGB B box or fragments thereof, orcells expressing the B box or epitope-bearing fragments can be used asan immunogen to produce antibodies immunospecific for the immunogen.“Antibodies” as used herein includes monoclonal and polyclonalantibodies, chimeric, single chain, simianized antibodies and humanizedantibodies, as well as Fab fragments, including the products of an Fabimmunoglobulin expression library.

Because all vertebrate HMGB B boxes show a high degree of sequenceconservation, it is believed that any vertebrate HMGB B box can inducerelease of a proinflammatory cytokine from a vertebrate cell. Therefore,antibodies against any vertebrate HMGB B box are within the scope of theinvention. Preferably, the HMGB B box is a mammalian HMGB B box, morepreferably a mammalian HMGB1 B box, most preferably a human HMGB1 B box,provided herein as SEQ ID NO:5, SEQ ID NO:20, or SEQ ID NO:58.Antibodies can also be directed against an HMGB B box fragment that hasB box biological activity.

Antibodies generated against the B box immunogen can be obtained byadministering the B box, a B box fragment, or cells comprising the B boxor B box fragment to an animal, preferably a nonhuman, using routineprotocols. The polypeptide, such as an antigenically or immunologicallyequivalent derivative or a fusion protein thereof is used as an antigento immunize a mouse or other animal such as a rat or chicken. The B boxor fragment immunogen can be provided as a fusion protein to providestability or increase the immunogenicity of the B box or fragment. Theimmunogen may be associated, for example, by conjugation, with animmunogenic carrier protein, for example, bovine serum albumin (BSA) orkeyhole limpet haemocyanin (KLH). Alternatively a multiple antigenicpeptide comprising multiple copies of the B box or fragment, may besufficiently antigenic to improve immunogenicity so as to obviate theuse of a carrier. Bispecific antibodies, having two antigen bindingdomains where each is directed against a different B box epitope, mayalso be produced by routine methods.

For preparation of monoclonal antibodies, any technique known in the artthat provides antibodies produced by continuous cell line cultures canbe used. See, e.g., Kohler and Milstein, Nature 256: 495-497, 1975;Kozbor et al., Immunology Today 4:72, 1983; and Cole et al., pg. 77-96in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., 1985.

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies to the Bbox or fragments. Also, transgenic mice, or other organisms such asother mammals, may be used to express humanized antibodies.

If the antibody is used therapeutically in in vivo applications, theantibody is preferably modified to make it less immunogenic in theindividual. For example, if the individual is human the antibody ispreferably “humanized”; where the complementarity determining region(s)of the antibody is transplanted into a human antibody (for example, asdescribed in Jones et al., Nature 321:522-525, 1986; and Tempest et al.,Biotechnology 9:266-273, 1991).

Phage display technology can also be utilized to select antibody geneswith binding activities towards the polypeptide either from repertoiresof PCR amplified v-genes of lymphocytes from humans screened forpossessing anti-B box antibodies or from naive libraries (McCafferty etal., Nature 348:552-554, 1990; and Marks, et al., Biotechnology10:779-783, 1992). The affinity of these antibodies can also be improvedby chain shuffling (Clackson et al., Nature 352: 624-628, 1991).

When the antibodies are obtained that specifically bind to HMGB B boxepitopes, they can then be screened without undue experimentation forthe ability to inhibit release of a proinflammatory cytokine.

Anti-HMGB B box antibodies that can inhibit the production of any singleproinflammatory cytokine are within the scope of the present invention.Preferably, the antibodies can inhibit the production of TNF, IL-1β, orIL-6. Most preferably, the antibodies can inhibit the production of anyproinflammatory cytokines produced by the vertebrate cell.

For methods of inhibiting release of a proinflammatory cytokine from acell or treating a condition characterized by activation of aninflammatory cytokine cascade using antibodies to the HMGB B box or abiologically active fragment thereof, the cell can be any cell that canbe induced to produce a proinflammatory cytokine. In preferredembodiments, the cell is an immune cell, for example, macrophages,monocytes, or neutrophils. In the most preferred embodiments, the cellis a macrophage.

In other embodiments, the present invention is directed to a compositioncomprising the antibody preparations described above, in apharmaceutically acceptable excipient. In these embodiments, thecompositions can inhibit a condition characterized by the activation ofan inflammatory cytokine cascade. Conditions that can be treated withthese compositions have been previously enumerated.

The antibody compositions described above can also include an antagonistof an early sepsis mediator, as previously described.

The B box polypeptides and biologically active fragments thereofdescribed in these embodiments can be used to induce inflammatorycytokines in the appropriate isolated cells in vitro, or ex vivo, or asa treatment in vivo. In any of these treatments, the polypeptide orfragment can be administered by providing a DNA or RNA vector encodingthe B box or B box fragment, with the appropriate control sequencesoperably linked to the encoded B box or B box fragment, so that the Bbox or B box fragment is synthesized in the treated cell or patient. Invivo applications include the use of the B box polypeptides or B boxfragment polypeptides or vectors as a weight loss treatment. See WO00/47104 (the entire teachings of which are incorporated herein byreference), demonstrating that treatment with HMGB1 induces weight loss.Since the HMGB B box has the activity of the HMGB protein, the B boxwould also be expected to induce weight loss. HMGB B box fragments thathave the function of the B box would also be expected to induce weightloss.

In further embodiments, the present invention is also directed to amethod of inhibiting the release of a proinflammatory cytokine from amammalian cell. The method comprises treating the cell with any of theHMGB A box compositions or any of the HMGB B box or HMGB B boxbiologically active fragment antibody compositions discussed above.

It is believed that this method would be useful for inhibiting thecytokine release from any mammalian cell that produces theproinflammatory cytokine. However, in preferred embodiments, the cell isa macrophage, because macrophage production of proinflammatory cytokinesis associated with several important diseases.

It is believed that this method is useful for the inhibition of anyproinflammatory cytokine produced by mammalian cells. In preferredembodiments, the proinflammatory cytokine is TNF, IL-1α, IL-1β, MIF orIL-6, because those proinflammatory cytokines are particularly importantmediators of disease.

The method of these embodiments is useful for in vitro applications,such as in studies for determining biological characteristics ofproinflammatory cytokine production in cells. However, the preferredembodiments are in vivo therapeutic applications, where the cells are ina patient suffering from, or at risk for, a condition characterized byactivation of an inflammatory cytokine cascade.

These in vivo embodiments are believed to be useful for any conditionthat is mediated by an inflammatory cytokine cascade, including any ofthose that have been previously enumerated. Preferred conditions includeappendicitis, peptic, gastric or duodenal ulcers, peritonitis,pancreatitis, ulcerative, pseudomembranous, acute or ischemic colitis,hepatitis, Crohn's disease, asthma, allergy, anaphylactic shock, organischemia, reperfusion injury, organ necrosis, hay fever, sepsis,septicemia, endotoxic shock, cachexia, septic abortion, disseminatedbacteremia, burns, Alzheimer's disease, cerebral infarction, cerebralembolism, spinal cord injury, paralysis, allograft rejection orgraft-versus-host disease. In the most preferred embodiments, thecondition is endotoxic shock or allograft rejection. Where the conditionis allograft rejection, the composition may advantageously also includean immunosuppressant that is used to inhibit allograft rejection, suchas cyclosporin.

These methods can also usefully include the administration of anantagonist of an early sepsis mediator. The nature of these antagonistshas been previously discussed.

In still other embodiments, the present invention is directed to amethod of treating a condition in a patient characterized by activationof an inflammatory cytokine cascade. The method comprises administeringto the patient with any of the HMGB A box compositions (includingnon-naturally occurring A box polypeptides and A box biologically activefragments) or any of the HMGB B box or B box biologically activefragment antibody compositions (including non-naturally occurring B boxpolypeptides or biologically active fragments thereof) discussed above.This method would be expected to be useful for any condition that ismediated by an inflammatory cytokine cascade, including any of thosethat have been previously enumerated. As with previously described invivo methods, preferred conditions include appendicitis, peptic, gastricor duodenal ulcers, peritonitis, pancreatitis, ulcerative,pseudomembranous, acute or ischemic colitis, hepatitis, Crohn's disease,asthma, allergy, anaphylactic shock, organ ischemia, reperfusion injury,organ necrosis, hay fever, sepsis, septicemia, endotoxic shock,cachexia, septic abortion, disseminated bacteremia, burns, Alzheimer'sdisease, cerebral infarction, cerebral embolism, spinal cord injury,paralysis, allograft rejection or graft-versus-host disease. In the mostpreferred embodiments, the condition is endotoxic shock or allograftrejection. Where the condition is allograft rejection, the compositionmay advantageously also include an immunosuppressant that is used toinhibit allograft rejection, such as cyclosporin.

These methods can also usefully include the administration of anantagonist of an early sepsis mediator. The nature of these antagonistshas been previously discussed.

In other embodiments, the present invention is directed to methods ofstimulating the release of a proinflammatory cytokine from a cell. Themethod comprises treating the cell with any of the B box polypeptides orbiologically active B box fragment polypeptides, for example, thesequence of SEQ ID NO:5, SEQ ID NO:20, SEQ ID NO:58, SEQ ID NO:16, orSEQ ID NO:23, as described herein (including non-naturally occurring Bbox polypeptides and fragments). This method is useful for in vitroapplications, for example, for studying the effect of proinflammatorycytokine production on the biology of the producing cell. The method isalso useful for in vivo applications, for example, in effecting weightloss or treating obesity in a patient, as previously discussed.

Thus, in additional embodiments, the present invention is directed to amethod for effecting weight loss or treating obesity in a patient. Themethod comprises administering to the patient an effective amount of anyof the B box polypeptides or B box fragment polypeptides describedherein (including non-naturally occurring B box polypeptides andfragments), in a pharmaceutically acceptable excipient.

Screening for Modulators of the Release of Proinflammatory Cytokinesfrom Cells

The present invention is also directed to a method of determiningwhether a compound (test compound) inhibits inflammation and/or aninflammatory response. The method comprises combining the compound with(a) a cell that releases a proinflammatory cytokine when exposed to avertebrate HMGB B box or a biologically active fragment thereof, and (b)the HMGB B box or a biologically active fragment thereof, thendetermining whether the compound inhibits the release of theproinflammatory cytokine from the cell, compared to a suitable control.A compound that inhibits the release of the proinflammatory cytokine inthis assay is a compound that can be used to treat inflammation and/oran inflammatory response. The HMGB B box or biologically active HMGB Bbox fragment can be endogenous to the cell or can be introduced into thecell using standard recombinant molecular biology techniques.

Any cell that releases a proinflammatory cytokine in response toexposure to a vertebrate HMGB B box or biologically active fragmentthereof in the absence of a test compound would be expected to be usefulfor this invention. It is envisioned that the cell that is selectedwould be important in the etiology of the condition to be treated withthe inhibitory compound that is being tested. For many conditions, it isexpected that the preferred cell is a human macrophage.

Any method for determining whether the compound inhibits the release ofthe proinflammatory cytokine from the cell would be useful for theseembodiments. It is envisioned that the preferred methods are the directmeasurement of the proinflammatory cytokine, for example, with any of anumber of commercially available ELISA assays. However, in someembodiments, the measurement of the inflammatory effect of releasedcytokines may be preferable, particularly when there are severalproinflammatory cytokines produced by the test cell. As previouslydiscussed, for many important disorders, the predominant proinflammatorycytokines are TNF, IL-1α, IL-1β, MIF or IL-6; particularly TNF.

The present invention also features a method of determining whether acompound increases an inflammatory response and/or inflammation. Themethod comprises combining the compound (test compound) with (a) a cellthat releases a proinflammatory cytokine when exposed to a vertebrateHMGB A box or a biologically active fragment thereof, and (b) the HMGB Abox or biologically active fragment, then determining whether thecompound increases the release of the proinflammatory cytokine from thecell, compared to a suitable control. A compound that decreases therelease of the proinflammatory cytokine in this assay is a compound thatcan be used to increase an inflammatory response and/or inflammation.The HMGB A box or HMGB A box biologically active fragment can beendogenous to the cell or can be introduced into the cell using standardrecombinant molecular biology techniques.

Similar to the cell types useful for identifying inhibitors ofinflammation, described above, any cell in which release of aproinflammatory cytokine is normally inhibited in response to exposureto a vertebrate HMGB A box or a biologically active fragment thereof inthe absence of any test compound would be expected to be useful for thisinvention. It is envisioned that the cell that is selected would beimportant in the etiology of the condition to be treated with theinhibitory compound that is being tested. For many conditions, it isexpected that the preferred cell is a human macrophage.

Any method for determining whether the compound increases the release ofthe proinflammatory cytokine from the cell would be useful for theseembodiments. It is envisioned that the preferred methods are the directmeasurement of the proinflammatory cytokine, for example, with any of anumber of commercially available ELISA assays. However, in someembodiments, the measurement of the inflammatory effect of releasedcytokines may be preferable, particularly when there are severalproinflammatory cytokines produced by the test cell. As previouslydiscussed, for many important disorders, the predominant proinflammatorycytokines are TNF, IL-1α, IL-1β, MIF or IL-6; particularly TNF.

Preferred embodiments of the invention are described in the followingexamples. Other embodiments within the scope of the invention will beapparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples and claims,be considered exemplary only.

Example 1 Materials and Methods

Cloning of HMGB1 and Production of HMGB1 Mutants

The following methods were used to prepare clones and mutants of humanHMGB1. Recombinant full length human HMGB1 (651 base pairs; GenBankAccession Number U51677) was cloned by PCR amplification from a humanbrain Quick-Clone cDNA preparation (Clontech, Palo Alto, Calif.) usingthe following primers; forward primer: 5′ GATGGGCAAAGGAGATCCTAAG 3′ (SEQID NO:6) and reverse primer: 5′ GCGGCCGCTTATTCATCATCATCATCTTC 3′ (SEQ IDNO:7). Human HMGB1 mutants were cloned and purified as follows. Atruncated form of human HMGB1 was cloned by PCR amplification from aHuman Brain Quick-Clone cDNA preparation (Clontech, Palo Alto, Calif.).The primers used were (forward and reverse, respectively):

Carboxy terminus mutant (557 bp): (SEQ ID NO: 8)5′ GATGGGCAAAGGAGATCCTAAG 3′ and (SEQ ID NO: 9) 5′ GCGGCCGCTCACTTGCTTTTTTCAGCCTTGAC 3′. Amino terminus + B box mutant (486 bp):(SEQ ID NO: 10) 5′ GAGCATAAGAAGAAGCACCCA 3′ and (SEQ ID NO: 11)5′ GCGGCCGC TCACTTGCTTTTTTCAGCCTTGAC 3′. B box mutant (233 bp): (SEQ IDNO: 12) 5′ AAGTTCAAGGATCCCAATGCAAAG 3′ and (SEQ ID NO: 13)5′ GCGGCCGCTCAATATGCAGCTATATCCTTTTC 3′. Amino terminus + A box mutant(261 bp): (SEQ ID NO: 13) 5′ GATGGGCAAAGGAGATCCTAAG 3′ and (SEQ ID NO:14) 5′ TCACTTTTTTGTCTCCCCTTTGGG 3′.

A stop codon was added to each mutant to ensure the accuracy of proteinsize. PCR products were subcloned into pCRII-TOPO vector EcoRI sitesusing the TA cloning method per manufacturer's instruction (Invitrogen,Carlsbad, Calif.). After amplification, the PCR product was digestedwith EcoRI and subcloned onto expression vector with a GST tag pGEX(Pharmacia); correct orientation and positive clones were confirmed byDNA sequencing on both strands. The recombinant plasmids weretransformed into protease deficient E. coli strains BL21 orBL21(DE3)plysS (Novagen, Madison, Wis.) and fusion protein expressionwas induced by isopropyl-D-thiogalactopyranoside (IPTG). Recombinantproteins were obtained using affinity purification with the glutathioneSepharose resin column (Pharmacia).

The HMGB mutants generated as described above have the following aminoacid sequences:

Wild type HMGB1: (SEQ ID NO: 18)MGKGDPKKPTGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRLPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAAKLKEKYEKDIAAYRAKGKPDAAKKGVVKAEKSKKKKEEEEDEEDEEDEEEE EDEEDEEDEEEDDDDECarboxy terminus mutant: (SEQ ID NO: 19)MGKGDPKKPTGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGETKKKFKDPNAPKRLPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAAKLKEKYEKDIAAYRAKGKPDAAKKGVVKAEKSK B Box mutant: (SEQ ID NO: 20)FKDPNAPKRLPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAAKLKEKYEKDIAAY Amino terminus + A Box mutant: (SEQ ID NO: 21)MGKGDPKKPTGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGET, wherein the A box consists of thesequence (SEQ ID NO: 22)PTGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGET

A polypeptide generated from a GST vector lacking HMGB1 protein wasincluded as a control (containing a GST tag only). To inactive thebacterial DNA that bound to the wild type HMGB1 and some of the mutants(carboxy terminus and B box), DNase I (Life Technologies), for carboxyterminus and B box mutants, or benzonase nuclease (Novagen, Madison,Wis.), for wild type HMGB1, was added at about 20 units/ml bacterialysate. Degradation of DNA was verified by ethidium bromide staining ofthe agarose gel containing HMGB1 proteins before and after thetreatment. The protein eluates were passed over a polymyxin B column(Pierce, Rockford, Ill.) to remove any contaminating LPS, and dialyzedextensively against phosphate buffered saline to remove excess reducedglutathione. The preparations were then lyophilized and redissolved insterile water before use. LPS levels were less than 60 pg/μg protein forall the mutants and 300 pg/μg for wild type HMG-1 as measured by Limulusamebocyte lysate assay (Bio Whittaker Inc., Walkersville, Md.). Theintegrity of protein was verified by SDS-PAGE. Recombinant rat HMGB1(Wang et al., Science 285: 248-251, 1999) was used in some experimentssince it does not have degraded fragments as observed in purified humanHMGB1.

Peptide Synthesis

Peptides were synthesized and HPLC purified at Utah State UniversityBiotechnology Center (Logan, Utah) at 90% purity. Endotoxin was notdetectable in the synthetic peptide preparations as measured by Limulusassay.

Cell Culture

Murine macrophage-like RAW 264.7 cells (American Type CultureCollection, Rockville, Md.) were cultured in RPMI 1640 medium (LifeTechnologies, Grand Island N.Y.) supplemented with 10% fetal bovineserum (Gemini, Catabasas, Calif.), penicillin and streptomycin (LifeTechnologies) and were used at 90% confluence in serum-free Opti-MEM Imedium (Life Technologies, Grand Island, N.Y.). Polymyxin B (Sigma, St.Louis, Mo.) was routinely added at 100-1,000 units/ml to neutralize theactivity of any contaminating LPS as previously described; polymyxin Balone did not influence cell viability assessed with trypan blue (Wanget al., supra). Polymyxin B was not used in experiments of syntheticpeptide studies.

Measurement of TNF Release from Cells

TNF release was measured by a standard murine fibroblast L929 (ATCC,American Type Culture Collection, Rockville, Md.) cytotoxicity bioassay(Bianchi et al., Journal of Experimental Medicine 183:927-936, 1996)with the minimum detectable concentration of 30 pg/ml. Recombinant mouseTNF was obtained from R&D system Inc., (Minneapolis, Minn.). Murinefibroblast L929 cells (ATCC) were cultured in DMEM (Life Technologies,Grand Island, N.Y.) supplemented with 10% fetal bovine serum (Gemini,Catabasas, Calif.), penicillin (50 units/ml) and streptomycin (50 μg/ml)(Life Technologies) in a humidified incubator with 5% CO₂.

Antibody Production

Polyclonal antibodies against HMGB1 B box were raised in rabbits(Cocalico Biologicals, Inc., Reamstown, Pa.) and assayed for titer byimmunoblotting. IgG was purified from anti-HMGB1 antiserum using ProteinA agarose according to manufacturer's instructions (Pierce, Rockford,Ill.). Anti-HMGB1 B box antibodies were affinity purified by usingcyanogen bromide activated Sepharose beads (Cocalico Biological, Inc.).Non-immune rabbit IgG was purchased from Sigma (St. Louis, Mo.).Antibodies detected full length HMGB1 and B box in immunoassay, but didnot cross react with TNF, IL-1 and IL-6.

Labeling of HMGB1 with Na-¹²⁵I and Cell Surface Binding

Purified HMGB1 protein (10 μg) was radiolabeled with 0.2 mCi ofcarrier-free ¹²⁵I (NEN Life Science products Inc., Boston, Mass.) usingIodo-beads (Pierce, Rockford, Ill.) according to the manufacturer'sinstructions. ¹²⁵I-HMGB1 protein was separated from un-reacted ¹²⁵I bygel chromatography columns (P6 Micro Bio-Spin Chromatography Columns,Bio-Rad Laboratories, Hercules, Calif.) previously equilibrated with 300mM sodium chloride, 17.5 mM sodium citrate, pH 7.0 and 0.1% bovine serumalbumin (BSA). The specific activity of the eluted HMGB1 was about2.8×10⁶ cpm/μg protein. Cell surface binding studies were performed aspreviously described (Yang et al., Am. J. Physiol. 275:C675-C683, 1998).RAW 264.7 cells were plated on 24-well dishes and grown to confluence.Cells were washed twice with ice-cold PBS containing 0.1% BSA andbinding was carried out at 4° C. for 2 hours with 0.5 ml binding buffercontaining 120 mM sodium chloride, 1.2 mM magnesium sulfate, 15 mMsodium acetate, 5 mM potassium chloride, 10 mM Tris.HCl, pH 7.4, 0.2%BSA, 5 mM glucose and 25,000 cpm ¹²⁵I-HMGB1. At the end of theincubation the supernatants were discarded and the cells were washedthree times with 0.5 ml ice-cold PBS with 0.1% BSA and lysed with 0.5 mlof 0.5 N NaOH and 0.1% SDS for 20 minutes at room temperature. Theradioactivity in the lysate was then measured using a gamma counter.Specific binding was determined as total binding minus the radioactivityobtained in the presence of an excess amount of unlabeled HMGB1 or A boxproteins.

Animal Experiments

TNF knock out mice were obtained from Amgen (Thousand Oaks, Calif.) andwere on a B6x129 background. Age-matched wild-type B6x129 mice were usedas control for the studies. Mice were bred in-house at the University ofFlorida specific pathogen-free transgenic mouse facility (Gainesville,Fla.) and were used at 6-8 weeks of age.

Male 6-8 week old Balb/c and C3H/HeJ mice were purchased from HarlenSprague-Dawley (Indianapolis, Ind.) and were allowed to acclimate for 7days before use in experiments. All animals were housed in the NorthShore University Hospital Animal Facility under standard temperature,and a light and dark cycle.

Cecal Ligation and Puncture

Cecal ligation and puncture (CLP) was performed as described previously(Fink and Heard, J. Surg. Res. 49:186-196, 1990; Wichmann et al., Crit.Care Med. 26:2078-2086, 1998; and Remick et al., Shock 4:89-95, 1995).Briefly, Balb/c mice were anesthetized with 75 mg/kg ketamine (FortDodge, Fort Dodge, Iowa) and 20 mg/kg of xylazine (Bohringer Ingelheim,St. Joseph, Mo.) intramuscularly. A midline incision was performed, andthe cecum was isolated. A 6-0 prolene suture ligature was placed at alevel 5.0 mm from the cecal tip away from the ileocecal valve.

The ligated cecal stump was then punctured once with a 22-gauge needle,without direct extrusion of stool. The cecum was then placed back intoits normal intra-abdominal position. The abdomen was then closed with arunning suture of 6-0 prolene in two layers, peritoneum and fasciaseparately to prevent leakage of fluid. All animals were resuscitatedwith a normal saline solution administered sub-cutaneously at 20 ml/kgof body weight. Each mouse received a subcutaneous injection of imipenem(0.5 mg/mouse) (Primaxin, Merck & Co., Inc., West Point, Pa.) 30 minutesafter the surgery. Animals were then allowed to recuperate. Mortalitywas recorded for up to 1 week after the procedure; survivors werefollowed for 2 weeks to ensure no late mortalities had occurred.

D-Galactosamine Sensitized Mice

The D-galactosamine-sensitized model has been described previously(Galanos et al., Proc Natl. Acad. Sci. USA 76: 5939-5943, 1979; andLehmann et al., J. Exp. Med. 165: 657-663, 1997). Mice were injectedintraperitoneally with 20 mg D-galactosamine-HCL (Sigma)/mouse (in 200μl PBS) and 0.1 or 1 mg of either HMGB1 B box or vector protein (in 200μl PBS). Mortality was recorded daily for up to 72 hours afterinjection; survivors were followed for 2 weeks, and no later deaths fromB box toxicity were observed.

Spleen Bacteria Culture

Fourteen mice received either anti-HMGB1 antibody (n=7) or control (n=7)at 24 and 30 hours after CLP, as described herein, and were euthanizedfor necropsy. Spleen bacteria were recovered as described previously(Villa et al., J. Endotoxin Res. 4:197-204, 1997). Spleens were removedusing sterile technique and homogenized in 2 ml PBS. After serialdilutions with PBS, the homogenate was plated as 0.15 ml aliquots ontryptic soy agar plates (Difco, Detroit, Mich.) and CFU were countedafter overnight incubation at 37° C.

Statistical Analysis

Data are presented as mean±SEM unless otherwise stated. Differencesbetween groups were determined by two-tailed Student's t-test, one-wayANOVA followed by the least significant difference test or 2 tailedFisher's Exact Test.

Example 2 Mapping the HMGB1 Domains for Promotion of Cytokine Activity

HMGB1 has 2 folded DNA binding domains (A and B boxes) and a negativelycharged acidic carboxyl tail). To elucidate the structural basis ofHMGB1 cytokine activity, and to map the inflammatory protein domain, weexpressed full length and truncated forms of HMGB1 by mutagenesis andscreened the purified proteins for stimulating activity in monocytecultures (FIG. 1). Full length HMGB1, a mutant in which the carboxyterminus was deleted, a mutant containing only the B box, and a mutantcontaining only the A box were generated. These mutants of human HMGB1were made by polymerase chain reaction (PCR) using specific primers asdescribed herein, and the mutant proteins were expressed using aglutathione S-transferase (GST) gene fusion system (Pharmacia Biotech,Piscataway, N.J.) in accordance with the manufacturer's instructions.Briefly, DNA fragments, made by PCR methods, were fused to GST fusionvectors and amplified in E. coli. The expressed HMGB1 protein and HMGB1mutants and were then isolated using GST affinity column.

The effect of the mutants on TNF release from Murine macrophage-like RAW264.7 cells (ATCC) was carried out as follows. RAW 264.7 cells werecultured in RPMI 1640 medium (Life Technologies, Grand Island N.Y.)supplemented with 10% fetal bovine serum (Gemini, Catabasas, Calif.),penicillin and streptomycin (Life Technologies). Polymyxin (Sigma, St.Louis, Mo.) was added at 100 units/ml to suppress the activity of anycontaminating LPS. Cells were incubated with 1 μg/ml of full length(wild-type) HMGB1 and each HMGB1 mutant protein in Opti-MEM I medium for8 hours, and conditioned supernatants (containing TNF which had beenreleased from the cells) were collected and TNF released from the cellswas measured by a standard murine fibroblast L929 (ATCC) cytotoxicitybioassay (Bianchi et al., supra) with the minimum detectableconcentration of 30 pg/ml. Recombinant mouse TNF was obtained from R & DSystems Inc., (Minneapolis, Minn.) and used as control in theseexperiments. The results of this study are shown in FIG. 1. Data in FIG.1 are all presented as mean±SEM unless otherwise indicated. (N=6-10).

As shown in FIG. 1, wild-type HMGB1 and carboxyl-truncated HMGB1significantly stimulated TNF release by monocyte cultures (murinemacrophage-like RAW 264.7 cells). The B box was a potent activator ofmonocyte TNF release. This stimulating effect of the B box was specific,because A box only weakly activated TNF release.

Example 3 HMGB1 B Box Protein Promotes Cytokine Activity in a DoseDependent Manner

To further examine the effect of HMGB1 B box on cytokine production,varying amounts of HMGB1 B box were evaluated for the effects on TNF,IL-1B, and IL-6 production in murine macrophage-like RAW 264.7 cells.RAW 264.7 cells were stimulated with B box protein at 0-10 μg/ml, asindicated in FIGS. 2A-2C for 8 hours. Conditioned media were harvestedand measured for TNF, IL-1β and IL-6 levels. TNF levels were measured asdescribed herein, and IL-11 and IL-6 levels were measured using themouse IL-1β and IL-6 enzyme-linked immunosorbent assay (ELISA) kits (R&DSystem Inc., Minneapolis, Minn.) and N>5 for all experiments. Theresults of the studies are shown in FIGS. 2A-2C.

As shown in FIG. 2A, TNF release from RAW 264.7 cells increased withincreased amounts of B box administered to the cells. As shown in FIG.2B, addition of 1 μg/ml or 10 μg/ml of B box resulted in increasedrelease of IL-1β from RAW 264.7 cells. In addition, as shown in FIG. 2C,IL-6 release from RAW 264.7 cells increased with increased amounts of Bbox administered to the cells.

The kinetics of B box-induced TNF release was also examined. TNF releaseand TNF mRNA expression was measured in RAW 264.7 cells induced by B boxpolypeptide or GST tag polypeptide only used as a control (vector) (10μg/ml) for 0 to 48 hours. Supernatants were analyzed for TNF proteinlevels by an L929 cytotoxicity assay (N=3-5) as described herein. FormRNA measurement, cells were plated in 100 mm plate and treated inOpti-MEM I medium containing B box polypeptide or the vector alone for0, 4, 8, or 24 hours, as indicated in FIG. 2D. The vector only samplewas assayed at the 4 hour time point. Cells were scraped off the plateand total RNA was isolated by RNAzol B method in accordance with themanufacturer's instructions (Tel-Test “B”, Inc., Friendswood, Tex.). TNF(287 bp) was measured by RNase protection assay (Ambion, Austin, Tex.).Equal loading and the integrity of RNA was verified by ethidium bromidestaining of the RNA sample on agarose-formaldehyde gel. The results ofthe RNase protection assay are shown in FIG. 2D. As shown in FIG. 2D, Bbox activation of monocytes occurred at the level of gene transcription,because TNF mRNA was increased significantly in monocytes exposed to Bbox protein (FIG. 2B). TNF mRNA expression was maximal at 4 hours anddecreased at 8 and 24 hours. The vector only control (GST tag) showed noeffect on TNF mRNA expression. A similar study was carried out measuringTNF protein released from RAW 264.7 cells 0, 4, 8, 24, 32 or 48 hoursafter administration of B box or vector only (GST tag), using the L929cytotoxicity assay described herein. Compared to the control (mediumonly), B box treatment stimulated TNF protein expression (FIG. 2F) andvector alone (FIG. 2E) did not. Data are representative of threeseparate experiments. Together these data indicate that the HMGB1 B boxdomain has cytokine activity and is responsible for the cytokinestimulating activity of full length HMGB1.

In summary, the HMGB1 B box dose-dependently stimulated release of TNF,IL-1β and IL-6 from monocyte cultures (FIGS. 2A-2C), in agreement withthe inflammatory activity of full length HMGB1 (Andersson et al., J.Exp. Med. 192: 565-570, 2000). In addition, these studies indicate thatmaximum TNF protein release occurred within 8 hours (FIG. 2F). Thisdelayed pattern of TNF release is similar to TNF release induced byHMGB1 itself, and is significantly later than the kinetics of TNFinduced by LPS (Andersson et al., supra).

Example 4 The First 20 Amino Acids of the HMGB1 B Box Stimulate TNFActivity

The TNF-stimulating activity of the HMGB1 B box was further mapped. Thisstudy was carried out as follows. Fragments of the B box were generatedusing synthetic peptide protection techniques, as described herein. FiveHMGB1 B box fragments (from SEQ ID NO:20), containing amino acids 1-20,16-25, 30-49, 45-64, or 60-74 of the HMGB1 B box were generated, asindicated in FIG. 3. RAW 264.7 cells were treated with B box (1 μg/ml)or a synthetic peptide fragment of the B box (10 μg/ml), as indicated inFIG. 3 for 10 hours and TNF release in the supernatants was measured asdescribed herein. Data shown are mean±SEM, (n=3 experiments, each donein duplicate and validated using 3 separate lots of synthetic peptides).As shown in FIG. 3, TNF-stimulating activity was retained by a syntheticpeptide corresponding to amino acids 1-20 of the HMGB1 B box of SEQ IDNO:20 (fkdpnapkrlpsafflfcse; SEQ ID NO:20). The TNF stimulating activityof the 1-20-mer was less potent than either the full length synthetic Bbox (1-74-mer), or full length HMGB1, but the stimulatory effects werespecific because the synthetic 20-mers for amino acid fragmentscontaining 16-25, 30-49, 45-64, or 60-74 of the HMGB1 B box did notinduce TNF release. These results are direct evidence that themacrophage stimulating activity of the B box specifically maps to thefirst 20 amino acids of the HMGB B box domain of SEQ ID NO:20). This Bbox fragment can be used in the same manner as a polypeptide encoding afull length B box polypeptide, for example, to stimulate releases of aproinflammatory cytokine, or to treat a condition in a patientcharacterized by activation of an inflammatory cytokine cascade.

Example 5 HMGB1 A Box Protein Antagonizes HMGB1 Induced CytokineActivity in a Dose Dependent Manner

Weak agonists are by definition antagonists. Since the HMGB1 A box onlyweakly induced TNF production, as shown in FIG. 1, the ability of HMGB1A box to act as an antagonist of HMGB1 activity was evaluated. Thisstudy was carried out as follows. Sub-confluent RAW 264.7 cells in24-well dishes were treated with HMGB1 (1 μg/ml) and 0, 5, 10, or 25μg/ml of A box for 16 hours in Opti-MEM I medium in the presence ofpolymyxin B (100 units/ml). The TNF-stimulating activity (assayed usingthe L929 cytotoxicity assay described herein) in the sample receiving noA box was expressed as 100%, and the inhibition by A box was expressedas percent of HMGB1 alone. The results of the effect of A box on TNFrelease from RAW 264.7 cells is shown in FIG. 4A. As shown in FIG. 4A,the A box dose-dependently inhibited HMGB1 induced TNF release with anapparent EC₅₀ of approximately 7.5 μg/ml. Data in FIG. 4A are presentedas mean±SD (n=2-3 independent experiments).

Example 6 HMGB1 A Box Protein Inhibits Full Length HMGB1 and HMGB1 B BoxCytokine Activity

Antagonism of full length HMGB1 activity by HMGB1 A box or GST tag(vector control) was also determined by measuring TNF release from RAW264.7 macrophage cultures stimulated by co-addition of A box with fulllength HMGB1. RAW 264.7 macrophage cells (ATCC) were seeded into 24-welltissue culture plates and used at 90% confluence. The cells were treatedwith HMGB1, and/or A boxes as indicated for 16 hours in Optimum I medium(Life Technologies, Grand Island, N.Y.) in the presence of polymyxin B(100 units/ml, Sigma, St. Louis, Mo.) and supernatants were collectedfor TNF measurement (mouse ELISA kit from R&D System Inc, Minneapolis,Minn.). TNF-inducing activity was expressed as a percentage of theactivity achieved with HMG-1 alone. The results of these studies areshown in FIG. 4B. FIG. 4B is a histogram of the effect of HMGB1, alone,A box alone, Vector (control) alone, HMGB1 in combination with A box,and HMGB1 in combination with vector. As shown in FIG. 4B, HMGB1 A boxsignificantly attenuated the TNF stimulating activity of full lengthHMGB1.

Example 7 HMGB1 A Box Protein Inhibits HMGB1 Cytokine Activity byBinding to it

To determine whether the HMGB1 A box acts as an antagonist by displacingHMGB1 binding, ¹²⁵I-labeled-HMGB1 was added to macrophage cultures andbinding was measured at 4° C. after 2 hours. Binding assays in RAW 264.7cells were performed as described herein. ¹²⁵I-HMGB1 binding wasmeasured in RAW 264.7 cells plated in 24-well dishes for the timesindicated in FIG. 5A. Specific binding shown equals totalcell-associated ¹²⁵I-HMGB1 (CPM/well) minus cell associated CPM/well inthe presence of 5,000 fold molar excess of unlabeled HMGB1. FIG. 5A is agraph of the binding of ¹²⁵I-HMGB1 over time. As shown in FIG. 5A, HMGB1exhibited saturable first order binding kinetics. The specificity ofbinding was assessed as described in Example 1.

In addition, ¹²⁵I-HMG-1 binding was measured in RAW 264.7 cells platedon 24-well dishes and incubated with ¹²⁵I HMGB1 alone or in the presenceof unlabeled HMGB1 or A box. The results of this binding assay are shownin FIG. 5B. Data represents mean±SEM from 3 separate experiments. FIG.5B is a histogram of the cell surface binding of ¹²⁵I-HMGB1 in theabsence of unlabeled HMGB1 or HMGB1 (HMGB1) A box, or in the presence of5,000 molar excess of unlabeled HMGB1 or HMGB1 A box, measured as apercent of the total CPM/well. In FIG. 5B, “Total” equals counts perminutes (CPM)/well of cell associated ¹²⁵I-HMGB1 in the absence ofunlabeled HMGB1 or A box for 2 hours at 4° C. “HMGB I” or “A box” equalsto CPM/well of cell-associated ¹²⁵I-HMGB1 in the presence of 5,000 molarexcess of unlabeled HMGB1 or A box. The data are expressed as thepercent of total counts obtained in the absence of unlabeled HMGB1proteins (2,382,179 CPM/well). These results indicate that the HMGB1 Abox is a competitive antagonist of HMGB1 activity in vitro that inhibitsthe TNF-stimulating activity of HMGB1.

Example 8 Inhibition of Full Length HMGB1 and HMGB1 B Box CytokineActivity by Anti-B Box Polyclonal Antibodies

The ability of antibodies directed against the HMGB1 B box to modulatedthe effect of full length or HMGB1 B box was also assessed. Affinitypurified antibodies directed against the HMGB1 B box (B box antibodies)were generated as described herein and using standard techniques. Toassay the effect of the antibodies on HMGB1 or HMGB1 B box-induced TNFrelease from RAW 264.7 cells, sub-confluent RAW 264.7 cells in 24-welldishes were treated with HMG-1 (1 μg/ml) or HMGB1 B box (10 μg/ml) for10 hours with or without anti-B box antibody (25 μg/ml or 100 μg/mlantigen affinity purified, Cocalico Biologicals, Inc., Reamstown, Pa.)or non-immune IgG (25 μg/ml or 100 μg/ml; Sigma) added. TNF release fromthe RAW 264.7 cells was measured using the L929 cytotoxicity assay asdescribed herein. The results of this study are shown in FIG. 6, whichis a histogram of TNF released by RAW 264.7 cells administered nothing,1 μg/ml HMGB1, 1 μg/ml HMGB1 plus 25 μg/ml anti-B box antibody, 1 μg/mlHMGB1 plus 25 μg/ml IgG (control), 10 μg/ml B-box, 10 μg/ml B-box plus100 μg/ml anti-B box antibody or 10 μg/ml B-box plus 100 μg/ml IgG(control). The amount of TNF released from the cells induced by HMGB1alone (without addition of B box antibodies) was set as 100%, the datashown in FIG. 6 are the results of 3 independent experiments. As shownin FIG. 6, affinity purified antibodies directed against the HMGB1 B boxsignificantly inhibited TNF release induced by either full length HMGB1or the HMGB1 B box. These results indicate that such an antibody can beused to modulate HMGB1 function.

Example 9 HMGB1 B Box Protein is Toxic to D-galactosamine-sensitizedBalb/c Mice

To investigate whether the HMGB1 B box has cytokine activity in vivo, weadministered HMGB1 B box protein to unanesthetized Balb/c micesensitized with D-galactosamine (D-gal), a model that is widely used tostudy cytokine toxicity (Galanos et al., supra). Briefly, mice (20-25gram, male, Harlan Sprague-Dawley, Indianapolis, Ind.) wereintraperitoneally injected with D-gal (20 mg) (Sigma) and B box (0.1mg/ml/mouse or 1 mg/ml/mouse) or GST tag (vector; 0.1 mg/ml/mouse or 1mg/ml/mouse), as indicated in Table 1. Survival of the mice wasmonitored up to 7 days to ensure no late death occurred. The results ofthis study are shown in Table 1.

TABLE 1 Toxicity of HMGB1 B box on D-galactosamine-sensitized Balb/cMice Treatment Alive/total Control — 10/10 Vector 0.1 mg/mouse 2/2   1mg/mouse 3/3 B box 0.1 mg/mouse 6/6   1 mg/mouse  2/8* P < 0.01 versusvector alone as tested by Fisher's Exact Test

The results of this study showed that the HMGB1 B box was lethal toD-galactosamine-sensitized mice in a dose-dependent manner. In allinstances in which death occurred, it occurred within 12 hours.Lethality was not observed in mice treated with comparable preparationsof the purified GST vector protein devoid of B box.

Example 10 Histology of D-Galactosamine-Sensitized Balb/c Mice orC3H/HeJ Mice Administered HMGB1 B Box Protein

To further assess the lethality of the HMGB1 B box protein in vivo theHMGB1 B box was again administered to D-galactosamine-sensitized Balb/cmice. Mice (3 per group) received D-gal (20 mg/mouse) plus B box orvector (1 mg/mouse) intraperitoneally for 7 hours and were thensacrificed by decapitation. Blood was collected, and organs (liver,heart, kidney and lung) were harvested and fixed in 10% formaldehyde.Tissue sections were prepared with hematoxylin and eosin staining forhistological evaluation (Criterion Inc., Vancouver, Canada). The resultsof these studies are shown in FIGS. 7A-7J, which are scanned images ofhematoxylin and eosin stained kidney sections (FIG. 7A), myocardiumsections (FIG. 7C), lung sections (FIG. 7E), and liver sections (FIGS.7G and 7I) obtained from an untreated mouse and kidney sections (FIG.7B), myocardium sections (FIG. 7D), lung sections (FIG. 7F), and liversections (FIGS. 7H and 7J) obtained from mice treated with the HMGB1 Bbox. Compared to the control mice, B box treatment caused no abnormalityin kidneys (FIGS. 7A and 7B) and lungs (FIGS. 7E and 7F). The mice hadsome ischemic changes and loss of cross striation in myocardial fibersin the heart (FIGS. 7C and 7D as indicated by the arrow in FIG. 7D).Liver showed most of the damage by the B box as illustrated by activehepatitis (FIGS. 7G-7J). In FIG. 7J, hepatocyte dropouts are seensurrounded by accumulated polymorphonuclear leukocytes. The arrows inFIG. 7J point to the sites of polymorphonuclear accumulation (dotted) orapoptotic hepatocytes (solid). Administration of HMGB1 B box in vivoalso stimulated significantly increased serum levels of IL-6 (315+93 vs.20+7 pg/ml, B box vs. control, p<0.05) and IL-1β (15+3 vs. 4+1 pg/ml, Bbox vs. control, p<0.05).

Administration of B box protein to C3H/HeJ mice (which do not respond toendotoxin) was also lethal, indicating that HMGB1 B box is lethal in theabsence of LPS signal transduction. Hematoxylin and eosin stainedsections of lung and kidney collected 8 hours after administration of Bbox revealed no abnormal morphologic changes. Examination of sectionsfrom the heart however, revealed evidence of ischemia with loss of crossstriation associated with amorphous pink cytoplasm in myocardial fibers.Sections from liver showed mild acute inflammatory responses, with somehepatocyte dropout and apoptosis, and occasional polymorphonuclearleukocytes. These specific pathological changes were comparable to thoseobserved after administration of full length HMGB1 and confirm that theB box alone can recapitulate the lethal pathological response to HMGB1in vivo.

To address whether the TNF-stimulating activity of HMGB1 contributes tothe mediation of lethality by B box, we measured lethality in TNFknock-out mice (TNF-KO, Nowak et al., Am. J. Physiol. Regul. Integr.Comp. Physiol. 278: R1202-R1209, 2000) and the wild-type controls(B6x129 strain) sensitized with D-galactosamine (20 mg/mouse) andexposed to B box (1 mg/mouse, injected intraperitoneally). The B box washighly lethal to the wild-type mice (6 dead out of nine exposed) butlethality was not observed in the TNF-KO mice treated with B box (0 deadout of 9 exposed, p<0.05 v. wild type). Together with the data from theRAW 264.7 macrophage cultures, described herein, these data now indicatethat the B box of HMGB1 confers specific TNF-stimulating cytokineactivity.

Example 11 HMGB1 Protein Level is Increased in Septic Mice

To examine the role of HMGB1 in sepsis, we established sepsis in miceand measured serum HMGB1 using a quantitative immunoassay describedpreviously (Wang et al., supra). Mice were subjected to cecal ligationand puncture (CLP), a well characterized model of sepsis caused byperforating a surgically-created cecal diverticulum, that leads topolymicrobial peritonitis and sepsis (Fink and Heard, supra; Wichmann etal., supra; and Remick et al., supra). Serum levels of HMGB1 were thenmeasured (Wang et al., supra). FIG. 8 shows the results of this study ina graph that illustrates the levels of HMGB1 in mice 0 hours, 8 hours,18 hours, 24 hours, 48 hours, and 72 hours after subjection to CLP. Asshown in FIG. 8, serum HMGB1 levels were not significantly increased forthe first eight hours after cecal perforation, then increasedsignificantly after 18 hours (FIG. 8). Increased serum HMGB1 remained atelevated plateau levels for at least 72 hours after CLP, a kineticprofile that is quite similar to the previously described, delayed HMGB1kinetics in endotoxemia (Wang et al., supra). This temporal pattern ofHMGB1 release corresponded closely to the development of signs of sepsisin the mice. During the first eight hours after cecal perforation theanimals were observed to be mildly ill, with some diminished activityand loss of exploratory behavior. Over the ensuing 18 hours the animalsbecame gravely ill, huddled together in groups with piloerection, didnot seek water or food, and became minimally responsive to externalstimuli or being examined by the handler.

Example 12 Treatment of Septic Mice with HMGB1 A Box Protein IncreasesSurvival of Mice

To determine whether the HMGB1 A box can inhibit the lethality of HMGB1during sepsis, mice were subjected to cecal perforation and treated byadministration of A box beginning 24 hours after the onset of sepsis.CLP was performed on male Balb/c mice as described herein. Animals wererandomly grouped, with 15-25 mice per group. The HMGB1 A box (60 or 600μg/mouse each time) or vector (GST tag, 600 μg/mouse) alone wasadministered intraperitoneally twice daily for 3 days beginning 24 hoursafter CLP. Survival was monitored twice daily for up to 2 weeks toensure no late death occurred. The results of this study are illustratedin FIG. 9, which is a graph of the effect of vector (GST; control) 60μg/mouse or 600 μg/mouse on survival over time (*P<0.03 vs. control astested by Fisher's exact test). As shown in FIG. 9, administration ofthe HMGB1 A box significantly rescued mice from the lethal effects ofsepsis, and improved survival from 28% in the animals treated withprotein purified from the vector protein (GST) devoid of the A box, to68% in animals receiving A box (P<0.03 by Fischer's exact test). Therescuing effects of the HMGB1 A box in this sepsis model were A boxdose-dependent; animals treated with 600 μg/mouse of A box were observedto be significantly more alert, active, and to resume feeding behavioras compared to either controls treated with vector-derived preparations,or to animals treated with only 60 μg A box. The latter animals remainedgravely ill, with depressed activity and feeding for several days, andmost died.

Example 13 Treatment of Septic Mice with Anti-HMGB1 Antibody IncreasesSurvival of Mice

Passive immunization of critically ill septic mice with anti-HMGB1antibodies was also assessed. In this study, male Balb/c mice (20-25 gm)were subjected to CLP, as described herein. Affinity purified anti-HMGB1B box polyclonal antibody or rabbit IgG (as control) was administered at600 μg/mouse beginning 24 hours after the surgery, and twice daily for 3days. Survival was monitored for 2 weeks. The results of this study areshown in FIG. 10A which is a graph of the survival of septic micetreated with either a control antibody or an anti-HMGB1 antibody. Theresults show that anti-HMGB1 antibodies administered to the mice 24hours after the onset of cecal perforation significantly rescued animalsfrom death as compared to administration of non-immune antibodies(p<0.02 by Fisher's exact test). Within 12 hours after administration ofanti-HMGB1 antibodies, treated animals showed increased activity andresponsiveness as compared to controls receiving non-immune antibodies.Whereas animals treated with non-immune antibodies remained huddled, illkempt, and inactive, the treated animals improved significantly andwithin 48 hours resumed normal feeding behavior. Anti-HMGB1 antibodiesdid not suppress bacterial proliferation in this model, because weobserved comparable bacterial counts (CFU, the aerobic colony formingunits) from spleen 31 hours after CLP in the treated animals as comparedto animals receiving irrelevant antibodies (control bacteriacounts=3.5±0.9×10⁴ CFU/g; n=7). Animals were monitored for up to 2 weeksafterwards, and late deaths were not observed, indicating that treatmentwith anti-HMGB1 conferred complete rescue from lethal sepsis, and didnot merely delay death.

To our knowledge, no other specific cytokine-directed therapeutic is aseffective when administered so late after the onset of sepsis. Bycomparison, administration of anti-TNF actually increases mortality inthis model, and anti-MIF antibodies are ineffective if administered morethan 8 hours after cecal perforation (Remick et al, supra; and Calandraet al., Nature Med. 6:164-170, 2000). These data demonstrate that HMGB1can be targeted as late as 24 hours after cecal perforation in order torescue lethal cases of established sepsis.

In another example of the rescue of endotoxemic mice using anti-B boxantibodies, anti-HMGB1 B box antibodies were evaluated for their abilityto rescue LPS-induced septic mice. Male Balb/c mice (20-25 gm, 26 pergroup) were treated with an LD75 dose of LPS (15 mg/kg) injectedintraperitoneally (IP). Anti-HMGB1 B box or non-immune rabbit serum (0.3ml per mouse each time, IP) was given at time 0, +12 hours and +24 hoursafter LPS administration. Survival of mice was evaluated over time. Theresults of this study are shown in FIG. 10B, which is a graph of thesurvival of septic mice administered anti-HMGB1 B box antibodies ornon-immune serum. As shown in FIG. 10B, anti-HMGB1 B box antibodiesimproved survival of the septic mice.

Example 14 Inhibition of HMGB1 Signaling Pathway Using an Anti-RAGEAntibody

Previous data implicated RAGE as an HMGB1 receptor that can mediateneurite outgrowth during brain development and migration of smoothmuscle cells in wound healing (Hori et al. J. Biol. chem.270:25752-25761, 1995; Merenmies et al. J. Biol. Chem. 266:16722-16729,1991; and Degryse et al., J. Cell Biol. 152:1197-1206, 2001). Wemeasured TNF release in RAW 264.7 cultures stimulated with HMGB1 (1μg/ml), LPS (0.1 μg/ml), or HMGB1 B box (1 μg/ml) in the presence ofanti-RAGE antibody (25 μg/ml) or non-immune IgG (25 μg/ml). Briefly, thecells were seeded into 24-well tissue culture plates and used at 90%confluence. LPS (E. coli 0111:B4, Sigma, St. Louis, Mo.) was sonicatedfor 20 minutes before use. Cells were treated with HMGB1 (1 μg/ml), LPS(0.1 μg/ml), or HMGB1 B box (1 μg/ml) in the presence of anti-RAGEantibody (25 μg/ml) or non-immune IgG (25 μg/ml) as indicated in FIG.11A for 16 hours in serum-free Opti-MEM I medium (Life Technologies) andsupernatants were collected for TNF measurement using the L929cytotoxicity assay described herein. IgG purified polyclonal anti-RAGEantibody (Catalog No. sc-8230, N-16, Santa Cruz Biotech, Inc., SantaCruz, Calif.) was dialyzed extensively against PBS before use. Theresults of this study are shown in FIG. 1A, which is a histogram of theeffects of HMGB1, LPS, or HMGB1 B box in the presence of anti-RAGEantibodies or non-immune IgG (control) on TNF release from RAW 264.7cells. As shown in FIG. 11A, compared to non-immune IgG, anti-RAGEantibody significantly inhibited HMGB1 B box-induced TNF release. Thissuppression was specific, because anti-RAGE did not significantlyinhibit LPS-stimulated TNF release. Notably, the maximum inhibitoryeffect of anti-RAGE decreased HMG-1 signaling by only 40%, suggestingthat other signal transduction pathways may participate in HMGB1signaling.

To examine the effects of HMGB1 or HMGB1 B Box on the NF-kB-dependentELAM promoter, the following experiment was carried out. RAW 264.7macrophages were transiently co-transfected with an expression plasmidencoding a murine MyD 88-dominant-negative (DN) mutant (corresponding toamino acids 146-296), or empty vector, plus a luciferase reporterplasmid under the control of the NF-kB-dependent ELAM promoter, asdescribed by Means et al. (J. Immunol. 166:4074-4082, 2001). A portionof the cells were then stimulated with full-length HMBG1 (100 ng/ml), orpurified HMGB1 B box (10 μg/ml), for 5 hours. Cells were then harvestedand luciferase activity was measured, using standard methods. Alltransfections were performed in triplicate, repeated at least threetimes, and a single representative experiment is shown in FIG. 11B. Asshown in FIG. 11B, HMGB1 stimulated luciferase activity in samples thatwere not co-transfected with the MyD 88 dominant negative, and the levelof stimulation was decreased in samples that were co-transfected withthe MyD 88 dominant negative. This effect was also observed in samplesadministered HMGB B box.

The effect of HMGB1 or HMGB1 B box on NF-kB activation was alsoexamined. CHO reporter cell lines that constitutively express humanToll-like receptor 2 (TLR2) or Toll-like receptor 4 (TLR4) have beenpreviously described (Means et al., J. Immunology, 163:3920-3927, 1999).These reporter lines also contain a stably transfected ELAM-CD25reporter gene, and express human CD25 on their surface as a consequenceof NF-kB activation. CHO/TLR2 and CHO/TLR4 cells were stimulated withIL-1 (10 ng/ml), purified full-length HMG-1 (100 ng/ml), or purified Bbox (10 μg/ml) for 18 hours. Following stimulation, cells were stainedwith a PE-labeled anti-CD25 monoclonal antibody and surface expressionof CD25 was measured by flow cytometry. The results of this study areshown ib FIG. 1C. Data are expressed as the ratio (fold-activation) ofthe percent of CD25⁺ cells in unstimulated and stimulated cellpopulations that were gated to exclude the lowest 5% of cells based onmean FL1 fluorescence. In CHO/TLR4 cells, stimulation with each of HMGB1and HMGB1 B box resulted in decreased CD25 expression compared to theCHO/TLR2 samples.

The effect of anti-RAGE antibodies, anti-TLR2 antibodies, a combinationof anti-RAGE antibodies and anti-TLR2 antibodies or IgG, onHMG-1-mediated TNF release in RAW 264.7 cells was also determined. RAW264.7 cells were seeded into 24-well tissue culture plates and used at90% confluence. Cells were incubated with HMG-1 with or withoutanti-RAGE antibody (Cat# sc-8230, Santa Cruz Biotech Inc., Santa Cruz,Calif.), anti-TLR2 antibody (Affinity-purified polyclonal antibody, Cat# sc-12504, D17, Santa Cruz) or IgG (non-immune IgG, Sigma, St. Louis,Mo.) in Optimum I medium (Life Technologies, Grand Island, N.Y.) in thepresence of polymyxin B (100 units/ml, Sigma, St. Louis, Mo.) for 16hours. Antibodies were dialyzed against PBS to remove sodium azidebefore use. Conditioned media were collected and a TNF ELISA wasperformed, using standard ELISA methods. Data (n=3) were expressed as apercentage of the activity achieved with HMG-1 alone. The results ofthis study are shown in FIG. 11D. Both anti-RAGE and anti-TLR2antibodies significantly (*P<0.05) inhibited HMG-1-mediated TNF release.Combination of the 2 antibodies had additive effects in inhibiting TNFrelease whereas IgG was irrelevant.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method of treating a condition in a patient characterized by anincreased serum HMGB 1 level as compared to a suitable control, andactivation of an inflammatory cytokine cascade, comprising administeringto the patient a therapeutically effective amount of a polypeptide, anantagonist of TNF and a pharmaceutically acceptable excipient, whereinsaid polypeptide does not contain an HMGB B box, and wherein saidpolypeptide inhibits release of a proinflammatory cytokine from avertebrate cell and comprises an amino acid sequence selected from thegroup consisting of: i) an amino acid sequence having at least 80%sequence identity to SEQ ID NO:22; ii) an amino acid sequence having atleast 80% sequence identity to SEQ ID NO:57; iii) an amino acid sequencehaving at least 80% sequence identity to SEQ ID NO:4; iv) the amino acidsequence of SEQ ID NO:57; v) a biologically active fragment of SEQ IDNO:57; vi) the amino acid sequence of SEQ ID NO:22; vii) a biologicallyactive fragment of SEQ ID NO:22; and viii) the amino acid sequence ofSEQ ID NO:4.
 2. A method of treating a condition in a patientcharacterized by an increased serum HMGB 1 level as compared to asuitable control, and activation of an inflammatory cytokine cascade,comprising administering to the patient a therapeutically effectiveamount of a polypeptide and a pharmaceutically acceptable excipient,wherein said polypeptide inhibits release of a proinflammatory cytokinefrom a vertebrate cell and consists of an amino acid sequence selectedfrom the group consisting of: i) the amino acid sequence of SEQ IDNO:57; ii) a biologically active fragment of SEQ ID NO:57; iii) theamino acid sequence of SEQ ID NO:22; iv) a biologically active fragmentof SEQ ID NO:22; and v) the amino acid sequence of SEQ ID NO:4.
 3. Themethod of claim 2, further comprising administering an antagonist ofTNF.
 4. A method of treating a condition in a patient characterized byan increased serum HMGB 1 level as compared to a suitable control, andactivation of an inflammatory cytokine cascade, comprising administeringto the patient a therapeutically effective amount of a fusion proteinand a pharmaceutically acceptable excipient, wherein said fusion proteincomprises a polypeptide, wherein said fusion protein does not contain anHMGB B box, wherein said fusion protein inhibits release of aproinflammatory cytokine from a vertebrate cell, and wherein saidpolypeptide consists of an amino acid sequence selected from the groupconsisting of: i) the amino acid sequence of SEQ ID NO:22; ii) abiologically active fragment of SEQ ID NO:22; iii) the amino acidsequence of SEQ ID NO:57; iv) a biologically active fragment of SEQ IDNO:57; and v) the amino acid sequence of SEQ ID NO:4.
 5. A method ofinhibiting release of a proinflammatory cytokine from a mammalian cell,the method comprising treating the cell with a polypeptide comprising avertebrate high mobility group box protein (HMGB) A box or anon-naturally occurring HMGB A box which can inhibit release of aproinflammatory cytokine from a vertebrate cell treated with highmobility group box (HMGB) protein in an amount sufficient to inhibitrelease of the proinflammatory cytokine from the cell, wherein saidpolypeptide does not contain an HMGB B box, and wherein said polypeptidecomprises an amino acid sequence selected from the group consisting of:i) the amino acid sequence of SEQ ID NO:57; ii) a biologically activefragment of SEQ ID NO:57; iii) the amino acid sequence of SEQ ID NO:4;iv) the amino acid sequence of SEQ ID NO:22; v) a biologically activefragment of SEQ ID NO:22; vi) an amino acid sequence having at least 80%sequence identity to SEQ ID NO:57; vii) an amino acid sequence having atleast 80% sequence identity to SEQ ID NO:4; and viii) an amino acidsequence having at least 80% sequence identity to SEQ ID NO:22.
 6. Themethod of claim 5, wherein said polypeptide comprises the amino acidsequence of SEQ ID NO:57.
 7. The method of claim 5, wherein saidpolypeptide comprises a biologically active fragment of SEQ ID NO:57. 8.The method of claim 5, wherein said polypeptide comprises the amino acidsequence of SEQ ID NO:4.
 9. The method of claim 5, wherein saidpolypeptide comprises the amino acid sequence of SEQ ID NO:22.
 10. Themethod of claim 5, wherein said polypeptide comprises a biologicallyactive fragment of SEQ ID NO:22.
 11. The method of claim 5, wherein saidpolypeptide comprises an amino acid sequence having at least 80%sequence identity to SEQ ID NO:57.
 12. The method of claim 5, whereinsaid polypeptide comprises an amino acid sequence having at least 80%sequence identity to SEQ ID NO:4.
 13. The method of claim 5, whereinsaid polypeptide comprises an amino acid sequence having at least 80%sequence identity to SEQ ID NO:22.
 14. A method of treating a patientwith sepsis, comprising administering to the patient a therapeuticallyeffective amount of a polypeptide, an antagonist of TNF and apharmaceutically acceptable excipient, wherein said polypeptide does notcontain an HMGB B box, and wherein said polypeptide inhibits release ofa proinflammatory cytokine from a vertebrate cell and comprises an aminoacid sequence selected from the group consisting of: i) an amino acidsequence having at least 80% sequence identity to SEQ ID NO:22; ii) anamino acid sequence having at least 80% sequence identity to SEQ IDNO:57; iii) an amino acid sequence having at least 80% sequence identityto SEQ ID NO:4; iv) the amino acid sequence of SEQ ID NO:57; v) abiologically active fragment of SEQ ID NO:57; vi) the amino acidsequence of SEQ ID NO:22; vii) a biologically active fragment of SEQ IDNO:22; and viii) the amino acid sequence of SEQ ID NO:4.
 15. The methodof claim 14, wherein said polypeptide comprises an amino acid sequencehaving at least 80% sequence identity to SEQ ID NO:22.
 16. The method ofclaim 14, wherein said polypeptide comprises an amino acid sequencehaving at least 80% sequence identity to SEQ ID NO:57.
 17. The method ofclaim 14, wherein said polypeptide comprises an amino acid sequencehaving at least 80% sequence identity to SEQ ID NO:4.
 18. The method ofclaim 14, wherein said polypeptide comprises the amino acid sequence ofSEQ ID NO:57.
 19. The method of claim 14, wherein said polypeptidecomprises a biologically active fragment of SEQ ID NO:57.
 20. The methodof claim 14, wherein said polypeptide comprises the amino acid sequenceof SEQ ID NO:22.
 21. The method of claim 14, wherein said polypeptidecomprises a biologically active fragment of SEQ ID NO:22.
 22. The methodof claim 14, wherein said polypeptide comprises the amino acid sequenceof SEQ ID NO:4.
 23. A method of treating a patient with sepsis,comprising administering to the patient a therapeutically effectiveamount of a polypeptide and a pharmaceutically acceptable excipient,wherein said polypeptide inhibits release of a proinflammatory cytokinefrom a vertebrate cell and consists of an amino acid sequence selectedfrom the group consisting of: i) the amino acid sequence of SEQ IDNO:57; ii) a biologically active fragment of SEQ ID NO:57; iii) theamino acid sequence of SEQ ID NO:22; iv) a biologically active fragmentof SEQ ID NO:22; and v) the amino acid sequence of SEQ ID NO:4.
 24. Themethod of claim 23, further comprising administering an antagonist ofTNF.
 25. A method of treating a patient with sepsis, comprisingadministering to the patient a therapeutically effective amount of afusion protein and a pharmaceutically acceptable excipient, wherein saidfusion protein comprises a polypeptide, wherein said fusion protein doesnot contain an HMGB B box, wherein said fusion protein inhibits releaseof a proinflammatory cytokine from a vertebrate cell, and wherein saidpolypeptide consists of an amino acid sequence selected from the groupconsisting of: i) the amino acid sequence of SEQ ID NO:22; ii) abiologically active fragment of SEQ ID NO:22; iii) the amino acidsequence of SEQ ID NO:57; iv) a biologically active fragment of SEQ IDNO:57; and v) the amino acid sequence of SEQ ID NO:4.
 26. The method ofclaim 25, wherein said polypeptide consists of an amino acid sequenceselected from the group consisting of SEQ ID NO:22 and a biologicallyactive fragment of SEQ ID NO:22.
 27. The method of claim 25, whereinsaid polypeptide consists of an amino acid sequence selected from thegroup consisting of SEQ ID NO:57 and a biologically active fragment ofSEQ ID NO:57.
 28. The method of claim 25, wherein said polypeptideconsists of the amino acid sequence of SEQ ID NO:4.